The

Mechanisms

of

Codon

Reassignments

in

Mitochondrial

Genetic

Codes

Many

cases

of

nonstandard

genetic

codes

are

known

in

mitochondrial

genomes

We

carry

out

analysis

of

phylogeny

and

codon

usage

of

organisms

for

which

the

complete

mitochondrial

genome

is

available

and

we

determine

the

most

likely

mechanism

for

codon

reassignment

in

each

case

Reassignment

events

can

be

classified

according

to

the

gain-loss

framework

The

gain

represents

the

appearance

of

a

new

tRNA

for

the

reassigned

codon

or

the

change

of

an

existing

tRNA

such

that

it

gains

the

ability

to

pair

with

the

codon

The

loss

represents

the

deletion

of

a

tRNA

or

the

change

in

a

tRNA

so

that

it

no

longer

translates

the

codon

One

possible

mechanism

is

codon

disappearance

-LRB-

CD

-RRB-

where

the

codon

disappears

from

the

genome

prior

to

the

gain

and

loss

events

In

the

alternative

mechanisms

the

codon

does

not

disappear

In

the

unassigned

codon

mechanism

the

loss

occurs

first

whereas

in

the

ambiguous

intermediate

mechanism

the

gain

occurs

first

Codon

usage

analysis

gives

clear

evidence

of

cases

where

the

codon

disappeared

at

the

point

of

the

reassignment

and

also

cases

where

it

did

not

disappear

CD

is

the

probable

explanation

for

stop

to

sense

reassignments

and

a

small

number

of

reassignments

of

sense

codons

However

the

majority

of

sense-to-sense

reassignments

can

not

be

explained

by

CD

In

the

latter

cases

by

analysis

of

the

presence

or

absence

of

tRNAs

in

the

genome

and

of

the

changes

in

tRNA

sequences

it

is

sometimes

possible

to

distinguish

between

the

unassigned

codon

and

the

ambiguous

intermediate

mechanisms

We

emphasize

that

not

all

reassignments

follow

the

same

scenario

and

that

it

is

necessary

to

consider

the

details

of

each

case

carefully

Introduction

Distinguishing

Possible

Mechanisms

of

Codon

Reassignment

Now

that

many

complete

genomes

of

organisms

and

organelles

are

available

there

is

ample

evidence

that

the

genetic

code

is

not

as

universal

-LRB-

Knight

et

al.

Yokobori

et

al.

Santos

et

al.

-RRB-

as

previously

believed

-LRB-

Crick

-RRB-

Many

cases

are

now

known

where

a

codon

-LRB-

or

related

group

of

codons

-RRB-

has

been

reassigned

from

one

amino

acid

to

another

from

a

stop

to

an

amino

acid

or

from

an

amino

acid

to

a

stop

If

a

change

in

the

translation

system

occurs

in

an

organism

such

that

a

codon

is

reassigned

most

of

the

occurrences

of

this

codon

will

still

be

at

places

where

the

old

amino

acid

was

preferred

We

would

expect

the

changes

causing

the

codon

reassignment

to

be

strongly

disadvantageous

and

to

be

eliminated

by

selection

It

is

possible

for

mutations

to

cause

disappearance

of

the

codon

in

its

original

positions

and

reappearance

in

positions

where

the

new

amino

acid

is

preferred

Mutations

throughout

the

genome

are

required

for

it

to

readjust

to

the

change

in

the

genetic

code

The

problem

is

therefore

to

understand

how

codon

reassignments

can

become

fixed

in

a

population

despite

being

apparently

deleterious

in

the

intermediate

stage

before

the

genome

has

time

to

readjust

Various

mechanisms

have

been

proposed

to

explain

the

process

of

codon

reassignment

We

have

recently

shown

-LRB-

Sengupta

and

Higgs

-RRB-

that

these

mechanisms

can

be

described

within

a

framework

that

we

call

the

gain-loss

framework

Gain

refers

to

the

gain

of

a

new

tRNA

gene

that

is

able

to

translate

the

reassigned

codon

as

a

different

amino

acid

or

the

gain

of

function

of

an

old

tRNA

gene

-LRB-

e.g.

by

base

modification

in

the

anticodon

-RRB-

so

that

it

translates

the

reassigned

codon

in

addition

to

the

codons

it

previously

interacted

with

Loss

refers

to

the

deletion

of

an

existing

tRNA

for

the

reassigned

codon

or

the

loss

of

function

of

the

tRNA

so

that

it

can

no

longer

translate

this

codon

We

identified

four

mechanisms

in

the

gain-loss

framework

as

described

below

Codon

disappearance

-LRB-

CD

-RRB-

mechanism

This

was

originally

proposed

by

Osawa

and

Jukes

-LRB-

-RRB-

For

an

amino

acid

-LRB-

or

stop

-RRB-

with

more

than

one

codon

it

is

possible

for

all

occurrences

of

a

codon

to

be

replaced

by

synonymous

codons

so

that

the

first

codon

disappears

entirely

from

the

genome

After

this

the

gain

and

loss

in

the

translation

system

are

neutral

changes

that

do

not

affect

the

organism

After

the

gain

and

loss

occur

the

codon

may

reappear

in

the

genome

by

mutations

at

sites

where

the

new

amino

acid

is

preferred

The

distinguishing

element

of

this

mechanism

is

that

the

codon

disappears

first

and

the

gain

and

loss

occur

during

a

period

in

which

the

codon

is

absent

For

the

other

three

mechanisms

described

here

the

codon

does

not

need

to

disappear

before

the

change

Ambiguous

intermediate

-LRB-

AI

-RRB-

mechanism

This

was

proposed

by

Schultz

and

Yarus

-LRB-

-RRB-

They

argued

that

a

codon

does

not

need

to

disappear

in

order

to

be

reassigned

and

proposed

that

there

is

a

transient

period

when

the

codon

is

ambiguously

translated

as

two

distinct

amino

acids

In

terms

of

our

gain-loss

framework

this

corresponds

to

the

case

where

the

gain

occurs

before

the

loss

i.e.

there

are

two

different

tRNAs

specific

to

the

amibiguous

codon

during

the

intermediate

period

and

the

new

code

becomes

established

when

the

old

tRNA

is

lost

Unassigned

codon

-LRB-

UC

-RRB-

mechanism

This

mechanism

arises

as

a

natural

possibility

in

our

gain-loss

framework

-LRB-

Sengupta

and

Higgs

-RRB-

It

corresponds

to

the

case

where

the

loss

occurs

before

the

gain

There

is

an

intermediate

period

where

there

is

no

tRNA

available

that

can

efficiently

translate

the

codon

hence

we

say

the

codon

is

unassigned

The

new

code

becomes

established

when

the

gain

in

function

of

the

new

tRNA

occurs

and

the

codon

is

reassigned

to

the

new

amino

acid

If

a

codon

were

truly

unassigned

and

no

tRNA

could

translate

it

at

all

then

the

loss

of

the

original

tRNA

would

be

lethal

if

the

codon

had

not

previously

disappeared

However

several

cases

are

known

where

an

alternative

tRNA

is

able

to

translate

a

codon

-LRB-

albeit

less

efficiently

-RRB-

after

the

tRNA

that

was

specific

to

that

codon

has

been

deleted

-LRB-

Yokobori

et

al.

-RRB-

Deletion

of

tRNAs

appears

to

be

frequent

in

mitochondrial

genomes

and

we

argue

below

that

deletion

of

a

tRNA

is

the

primary

event

that

instigated

several

of

the

mitochondrial

codon

reassignments

Compensatory

change

mechanism

The

final

mechanism

that

occurs

in

the

gain-loss

framework

is

referred

to

as

compensatory

change

because

of

its

analogy

with

compensatory

mutations

in

molecular

evolution

Kimura

-LRB-

-RRB-

considered

a

pair

of

mutations

such

that

each

is

deleterious

when

it

occurs

alone

but

when

both

occur

together

they

are

neutral

e.g.

in

the

paired

regions

of

RNA

secondary

structures

-LRB-

Higgs

Savill

et

al.

-RRB-

The

gain

and

the

loss

in

codon

reassignment

are

changes

in

two

different

parts

of

a

genome

that

form

a

compensatory

pair

It

is

possible

that

one

of

these

changes

occurs

but

remains

infrequent

in

the

population

until

the

second

change

occurs

in

an

individual

that

already

has

the

first

change

Once

the

gain

and

loss

are

present

in

the

same

individual

they

can

spread

simultaneously

through

the

population

although

they

did

not

occur

at

the

same

time

In

the

compensatory

change

case

there

is

no

point

at

which

individuals

with

ambiguous

codons

or

UCs

are

frequent

in

the

population.We

showed

using

a

population

genetics

simulation

-LRB-

Sengupta

and

Higgs

-RRB-

that

all

four

mechanisms

can

occur

within

the

same

model

depending

on

the

parameter

values

Here

we

consider

which

mechanisms

occur

in

real

cases

We

limit

ourselves

to

mitochondrial

changes

because

most

of

the

observed

changes

are

in

mitochondria

and

because

the

availability

of

substantial

numbers

of

complete

mitochondrial

genomes

makes

it

possible

to

pinpoint

the

changes

and

to

study

codon

usage

and

tRNA

gene

content

in

the

genomes

before

and

after

the

reassignment

Studies

of

this

type

were

carried

out

several

years

ago

by

Knight

et

al.

-LRB-

-RRB-

and

more

recently

by

Swire

et

al.

-LRB-

-RRB-

We

compare

our

results

with

these

previous

surveys

in

the

discussion

section.In

order

to

interpret

the

changes

in

the

genetic

code

phylogenetic

trees

are

required

We

split

our

set

of

species

into

groups

that

are

consistent

with

previous

studies

Figure

shows

fungi

and

related

species

and

Fig.

shows

plants/algae

and

related

species

These

were

obtained

by

our

own

phylogenetic

analysis

of

mitochondrial

genes

Figure

shows

alveolates/stramenopiles

and

related

species

and

Fig.

shows

metazoa

These

were

obtained

using

combined

information

from

other

sources

Details

of

phylogenetic

methods

are

given

in

the

Supplementary

Information

Fig.

Phylogeny

of

fungi

and

related

species

derived

from

mitochondrial

proteins.Fig

Phylogeny

of

plants

and

algae

derived

from

mitochondrial

proteins.Fig

Phylogeny

of

alveolates

stramenopiles

and

haptophytes

according

to

published

sources.Fig

Phylogeny

of

metazoa

according

to

published

sources

Reassignments

That

Can

Be

Explained

by

Codon

Disappearance

Reassignments

of

UGA

from

Stop

to

Trp

The

UGA

Stop-to-Trp

change

is

the

most

frequently

occurring

reassignment

known

The

review

by

Knight

et

al.

-LRB-

-RRB-

lists

six

of

these

in

mitochondria

-LRB-

and

four

in

nuclear

genomes

-RRB-

Our

updated

analysis

of

the

mitochondrial

data

identifies

the

following

cases

in

mitochondria.Metazoa

Monosiga

and

Amoebidium

This

change

is

shared

by

all

Metazoa

and

by

their

two

closest

known

relatives

-LRB-

Fig.

and

Lang

et

al.

-RRB-

Acanthamoeba

Fig

and

Burger

et

al.

-LRB-

-RRB-

Basidiomycota

-LRB-

Crinipellis

and

Schizophyllum

-RRB-

Fig.

Ascomycota

-LRB-

group

containing

Penicillium

and

relatives

-RRB-

Fig.

Ascomycota

-LRB-

group

containing

Yarrowia

and

relatives

-RRB-

Knight

et

al.

-LRB-

-RRB-

list

a

single

change

in

the

ancestor

of

Metazoa

Acanthamoeba

and

Fungi

However

Fig.

shows

that

UGA

remains

a

stop

codon

in

Chytridiomycota

Zygomycota

and

Dictyostelium

Therefore

it

is

likely

that

cases

i

ii

and

iii

are

separate

reassignments

The

reassignment

has

also

occurred

in

almost

all

the

Ascomycota

with

the

exception

of

the

Schizosaccharomyces

group

This

implies

that

cases

iv

and

v

in

the

Ascomycota

are

also

separate

reassignments

These

conclusions

depend

on

the

argument

that

a

reversal

of

this

change

-LRB-

from

Trp

to

Stop

-RRB-

is

very

unlikely

which

we

discuss

below.Rhodophyta

-LRB-

Chondrus

Porphyra

-RRB-

Fig.

and

Burger

et

al.

-LRB-

-RRB-

Pedinomonas

Fig.

and

Turmel

et

al.

-LRB-

-RRB-

Haptophytes

This

change

was

reported

in

a

subgroup

of

haptophytes

including

Phaecocystis

and

Isochrisis

by

Hayashi-Ishimaru

et

al.

-LRB-

-RRB-

The

complete

genome

of

Emiliana

-LRB-

Sanchez-Puerta

et

al.

-RRB-

shows

that

it

also

possesses

the

reassignment

This

is

consistent

with

our

phylogeny

-LRB-

Fig.

-RRB-

Ciliates

-LRB-

Paramecium

Tetrahymena

-RRB-

Knight

et

al.

-LRB-

-RRB-

place

this

change

at

the

base

of

the

alveolates

However

our

analysis

of

codon

usage

and

sequences

from

Plasmodium

species

discussed

below

shows

that

UGA

remains

a

Stop

codon

in

Plasmodium

Thus

the

reassignment

is

not

shared

by

all

alveolates.Cafeteria

Fig.

Bacillariophyta

-LRB-

Skeletonema

Thalassiosira

-RRB-

Fig

and

Ehara

et

al.

-LRB-

-RRB-

Kinetoplastida

-LRB-

Trypanosoma

Leishmania

-RRB-

Inagaki

et

al.

-LRB-

-RRB-

-LRB-

not

shown

in

figures

-RRB-

In

species

in

which

only

the

UGG

codon

codes

for

Trp

the

tRNA-Trp

has

a

CCU

anticodon

Mutation

of

the

wobble

position

C

to

a

U

creates

a

UCU

anticodon

that

can

pair

with

both

UGA

and

UGG

hence

the

reassignment

of

UGA

to

Trp

We

have

analyzed

the

tRNA-Trp

sequences

of

all

available

mitochondrial

genomes

to

determine

in

which

species

this

mutation

has

occurred

We

find

that

all

species

in

which

UGA

is

Stop

have

a

CCU

anticodon

as

expected

Almost

all

species

in

which

UGA

is

Trp

have

a

UCU

anticodon

but

we

find

some

exceptions

below

Table

shows

the

number

of

occurrences

of

each

of

the

standard

Stop

codons

together

with

the

tRNA-Trp

anticodon

When

UGA

is

Stop

it

is

usually

used

less

frequently

than

the

preferred

stop

codon

UAA

When

the

reassignment

occurs

many

UGG

Trp

codons

mutate

synonymously

to

UGA

hence

UGA

becomes

frequent

This

is

seen

in

all

species

having

a

UCA

anticodon

However

UGA

is

also

frequent

in

Amoebidium

Crinipellis

and

Schizophyllum

which

have

a

CCA

anticodon

Therefore

there

must

be

a

posttranslational

modification

of

the

C

base

in

the

anticodon

that

permits

translation

of

UGA

rather

than

a

mutation

in

the

gene

Another

special

case

is

the

kinetoplastids

These

have

no

mitochondrial

tRNAs

and

import

all

the

required

tRNAs

from

the

nucleus

The

tRNA-Trp

from

the

nucleus

has

CCA

anticodon

because

the

canonical

code

is

used

in

the

nucleus

The

same

tRNA

is

imported

to

the

mitochondrion

and

then

undergoes

a

base

modification

so

that

it

can

translate

both

UGA

and

UGG

-LRB-

Alfonzo

et

al.

-RRB-

Table

Codon

usage

data

relevant

to

reassignments

of

Stop

codons

UGA

and

UAGUGA

istRNA-Trp

anticodonCodon

usageUGAUAAUAGSpecies

in

Fig.

Amoebidium

Dictyostelium

Monosiga

Homo

Rhizophidium

sp.

Spizellomyces

Hyaloraphidium

curvatumNot

Monoblepharella

sp.

JEL

Harpochytrium

sp.

Harpochytrium

sp.

Allomyces

macrogynusNot

Mortierella

Rhizopus

oryzaeNot

Crinipellis

Schizophyllum

Penicillium

Hypocrea

Schizosaccharomyces

japonicusNot

Schizosaccharomyces

octosporusNot

Schizosaccharomyces

Yarrowia

Candida

Candida

Saccharomyces

in

Fig.

Malawimonas

Cyanidioschyzon

Chondrus

Porphyra

Chaetosphaeridium

Chara

Prototheca

wickerhamiiNot

Pseudoendoclonium

Pedinomonas

Scenedesmus

Chlamydomonas

eugametosNot

Chlamydomonas

reinhardtiiNot

in

Fig.

Emiliana

Rhodomonas

Naegleria

gruberiNot

Plasmodium

reichenowiNot

Plasmodium

falciparumNot

Paramecium

Tetrahymena

Tetrahymena

Caferteria

Phytophthora

Saprolegnia

feraxNot

Chrysodidymus

synuroidesNot

Ochramonas

danicaNot

Laminaria

Pylaiella

species

have

a

CCA

anticodon

but

UGA

is

Trp

This

suggests

that

the

C

base

is

modified

in

the

tRNA.bIn

vertebrates

AGA

and

AGG

are

also

Stop

codons

Each

is

used

once

in

Homo

sapiens.cUAG

is

reassigned

to

Leu

in

these

species.dThese

codons

are

presumed

to

be

inefficiently

translated

as

Trp

by

the

tRNA-Trp

-LRB-

CCA

-RRB-

eIn

S.

obliquus

UCA

is

a

novel

Stop

codon

occurring

times

The

predictions

from

our

theory

and

simulations

-LRB-

Sengupta

and

Higgs

-RRB-

were

that

Stop

codons

are

most

likely

to

be

reassigned

via

the

CD

mechanism

because

they

are

rare

in

the

first

place

-LRB-

and

chance

disappearance

is

therefore

relatively

likely

-RRB-

and

also

because

if

the

codons

do

not

disappear

the

penalty

for

read-through

of

a

stop

codon

is

likely

to

be

larger

than

the

penalty

for

mistranslation

of

an

amino

acid

-LRB-

However

read-through

of

a

stop

codon

is

not

necessarily

lethal

especially

if

the

number

of

additional

codons

translated

until

the

next

random

occurrence

of

a

stop

codon

is

not

too

large

-RRB-

The

figures

in

Table

make

a

strong

case

that

the

CD

mechanism

is

responsible

UGA

is

rare

in

almost

all

species

where

it

is

used

as

a

Stop

codon

Many

of

these

genomes

have

high

AU

content

which

is

probably

the

reason

UAA

is

preferred

over

UGA

as

a

Stop

codon

Many

of

the

closest

relatives

to

the

species

where

UGA

is

reassigned

have

particularly

low

usage

of

UGA

e.g.

Allomyces

and

Rhizopus

close

relatives

of

the

Basidiomycota

have

zero

Cyanidioschyzon

a

close

relative

of

Chondrus

and

Porphyra

has

only

two

Scenedesmus

and

Chlamydomonas

close

relatives

of

Pedinomonas

have

one

and

zero

respectively

and

Plasmodium

a

close

relative

of

Paramecium

and

Tetrahymena

has

zero

This

last

example

shows

that

UGA

is

not

reassigned

in

Plasmodium

as

mentioned

in

case

ix

above

These

examples

clearly

show

that

disappearance

of

UGA

is

possible

and

that

in

many

of

the

cases

there

is

good

evidence

that

UGA

was

absent

or

almost

absent

at

the

time

it

was

reassigned

A

mutation

pressure

from

GC

to

AU

which

is

implicated

in

the

disappearance

of

UGA

will

also

tend

to

cause

rapid

mutations

from

UGG

Trp

codons

to

UGA

after

the

Trp

tRNA

gains

the

ability

to

decode

UGA

This

is

one

reason

that

reversal

of

the

change

is

unlikely

There

are

often

around

UGAs

in

genomes

where

the

reassignment

has

occurred

and

it

would

be

very

difficult

for

this

large

number

to

disappear

by

chance

because

this

would

act

against

the

mutation

pressure

A

second

reason

is

that

the

reassignment

to

Trp

would

be

associated

with

the

loss

of

function

of

the

release

factor

that

originally

interacted

with

the

UGA

A

reversal

would

also

require

regaining

of

the

function

of

the

release

factor

The

unlikeliness

of

the

reversal

of

the

reassignment

is

important

in

our

interpretation

of

cases

i

to

v

above

The

codon

usage

and

tRNA-Trp

anticodon

data

-LRB-

Table

-RRB-

show

that

UGA

is

not

established

as

a

Trp

codon

in

Dictyostelium

Chytridiomycota

Zygomycota

or

Schizosaccharomyces

This

means

that

multiple

reassignment

events

are

required

to

explain

the

observed

pattern

It

appears

that

UGA

was

rare

and

prone

to

disappearance

right

from

the

base

of

the

Metazoa/Fungi

tree

in

Fig.

and

the

release

factor

may

already

have

lost

its

function

Therefore

all

that

is

required

for

the

reassignment

to

occur

is

the

simple

mutation

of

the

tRNA

anticodon

In

groups

where

the

release

factor

was

lost

but

the

tRNA

mutation

did

not

occur

the

UGA

codon

would

be

effectively

unassigned

and

would

be

selected

against

Seif

et

al.

-LRB-

-RRB-

argue

that

this

has

occurred

in

Mortierella

and

Schizosaccharomyces

where

there

is

evidence

of

a

small

number

of

UGA

acting

as

Trp

codons

that

are

translated

very

inefficiently

by

the

standard

Trp

tRNA

with

CCA

anticodon

A

genetic

code

change

might

easily

become

established

in

these

species

in

the

future

if

the

mutation

occurred

in

the

tRNA

The

Probability

of

Disappearance

of

UGA

Codons

It

is

possible

to

calculate

the

probability

Pdis

that

a

codon

will

disappear

using

a

simple

model

of

the

mutation

process

Swire

et

al.

-LRB-

-RRB-

have

used

this

method

to

show

that

in

most

cases

of

stop

codon

reassignment

the

probability

of

disappearance

of

the

codon

was

relatively

large

whereas

in

many

cases

of

sense

codon

reassignments

the

probability

was

extremely

small

Hence

they

argue

that

stop

codons

were

reassigned

via

CD

but

sense

codons

were

not

We

agree

with

this

conclusion

in

almost

all

cases

but

there

are

a

few

cases

where

we

argue

for

CD

in

sense

codon

reassignments

as

well

In

this

section

we

consider

only

UGA

stop

codons

The

probability

of

UGA

CD

can

be

calculated

as

follows

Let

the

equilibrium

frequencies

of

the

bases

under

the

mutational

process

be

πA

πC

πG

and

πU

Let

fUGA

fUAA

and

fUAG

be

the

relative

frequencies

of

the

three

stop

codons

-LRB-

fUGA

fUAA

fUAG

-RRB-

If

there

is

no

selective

preference

of

one

stop

codon

over

another

we

expect

these

frequencies

to

be

in

equilibrium

under

mutation

Therefore

Hence

In

mitochondrial

genomes

the

two

strands

of

the

genome

are

not

equivalent

the

four

base

frequencies

are

all

different

and

it

is

not

true

that

πC

πG

and

πA

πU

-LRB-

see

Urbina

et

al.

-RRB-

The

values

of

the

frequencies

can

be

estimated

from

the

frequencies

of

the

bases

at

fourfold

degenerate

-LRB-

FFD

-RRB-

sites

These

are

calculated

by

summing

over

all

third-position

sites

that

are

FFD

If

the

total

number

of

stop

codons

in

the

genome

is

Nstop

then

the

probability

of

disappearance

of

UGA

is

We

do

not

know

the

values

of

Nstop

or

fUGA

at

the

point

where

the

codon

reassignment

occurred

but

we

can

use

the

species

that

are

close

to

the

reassignment

point

on

the

tree

as

an

estimate

as

shown

in

Table

Nstop

is

the

the

sum

of

UAA

and

UAG

from

Table

UGA

codons

do

not

contribute

to

Nstop

because

these

are

now

Trp

codons

in

these

species

We

used

Monosiga

as

a

proxy

for

case

i

The

resulting

probability

is

high

-LRB-

Pdis

-RRB-

and

is

consistent

with

the

CD

mechanism

Amoebidium

was

not

used

in

this

case

because

it

has

a

derived

multichromosome

structure

of

the

mitochondrial

genome

and

metazoa

were

not

used

because

they

have

a

much

reduced

genome

size

Both

of

these

would

be

poor

estimators

of

what

the

genome

was

like

at

the

reassignment

point

In

all

these

species

πG

is

much

less

than

πA

but

the

bias

is

more

extreme

in

some

species

than

others

In

case

ix

if

either

of

the

Tetrahymena

species

is

used

as

proxy

a

fairly

high

Pdis

is

obtained

but

if

Paramecium

is

used

Pdis

is

much

lower

Similarly

in

case

iii

Pdis

is

high

if

we

use

Schizophyllum

but

low

if

we

use

Crinipellis

These

two

species

differ

in

base

frequencies

and

also

in

the

number

of

genes

on

the

genome

-LRB-

i.e.

Nstop

-RRB-

Although

Nstop

in

Crinipellis

-LRB-

also

known

as

Moniliophthora

perniciosa

-RRB-

this

genome

has

many

genes

labeled

as

hypothetical

protein

that

do

not

have

homologues

in

related

species

These

genes

may

be

recent

insertions

or

may

not

even

be

expressed

sequences

Thus

Nstop

was

probably

much

less

than

at

the

time

of

the

reassignment

and

Pdis

would

be

correspondingly

higher

In

both

these

cases

it

is

reasonable

to

conclude

that

the

properties

of

the

genome

at

the

point

of

reassignment

were

such

that

CD

was

not

too

unlikely

Table

Estimates

of

stop

codon

disappearance

probabilitiesCaseSpeciesNstop

G

Afcodon

-LRB-

-RRB-

PdisUGA

CDiMonosiga

iiAcanthamoeba

castellanii409.8926.3521.446.4E-05iiiCrinipellis

perniciosa899.1932.8417.942.3E-08iiiSchizophyllum

ivPenicillium

ivHypocrea

vYarrowia

vCandida

viChondrus

crispus256.0836.6112.463.6E-02viPorphyra

purpurea3110.9835.8019.011.4E-03viiPedinomonas

viiiEmiliana

huxleyi219.2636.5016.832.1E-02ixParamecium

aurelia4614.8719.6030.146.8E-08ixTetrahymena

pyriformis443.2242.276.624.9E-02ixTetrahymena

thermophila452.6340.465.757.0E-02xCafeteria

CDiRhizophidium373.3334.058.184.3E-02iSpizellomyces

punctatus326.0330.6914.107.7E-03iiScenedesmus

The

only

other

case

where

Pdis

is

low

is

case

ii

where

Pdis

if

Acathamoeba

is

used

as

proxy

There

is

no

other

related

species

for

which

data

are

available

We

have

already

seen

that

fluctuations

in

base

frequencies

can

change

Pdis

by

several

orders

of

magnitude

Therefore

a

value

of

order

does

not

appear

ridiculously

small

and

the

true

value

could

well

have

been

much

higher

at

the

point

the

reassignment

actually

occurred

Finally

we

comment

on

case

of

Swire

et

al.

Table

where

they

estimate

the

low

probability

of

Pdis

This

corresponds

to

our

case

vi

where

we

estimate

Pdis

if

Chondrus

is

used

as

proxy

and

Pdis

if

Porphyra

is

used

Both

of

these

are

much

higher

than

the

estimate

of

Swire

et

al.

We

also

note

that

according

to

our

phylogeny

these

two

species

are

related

and

we

assume

a

single

reassignment

in

their

ancestor

whereas

Swire

et

al.

treat

them

as

independent

-LRB-

cases

and

-RRB-

The

change

in

the

phylogeny

would

not

affect

our

estimate

of

Pdis

Thus

we

conclude

that

all

these

examples

of

UGA

codon

reassignment

are

consistent

with

the

CD

mechanism

-LRB-

Cases

xi

and

xii

are

not

included

in

Table

because

there

are

insufficient

data

to

perform

the

calculation

-RRB-

Reassignments

of

UAG

Stop

Codons

Two

cases

of

reassignment

of

UAG

from

Stop

to

Leu

are

known

one

in

the

two

chytrids

Rhyzophidium

and

Spizellomyces

-LRB-

Fig.

Laforest

et

al.

-RRB-

and

the

other

in

Scenedesmus

-LRB-

Fig.

Hayashi-Ishimaru

et

al.

Kück

et

al.

-RRB-

In

Table

UAG

is

also

seen

to

be

rare

in

general

and

rare

particularly

in

the

close

relatives

of

the

species

reassigned

only

two

in

the

other

chytrid

Hyaloraphidium

and

zero

in

Pedinomonas

relative

of

Scenedesmus

These

reassignments

show

the

same

pattern

as

the

UGA

Stop-to-Trp

examples

and

can

also

be

attributed

to

the

CD

mechanism

Disappearance

of

UAG

would

also

be

favored

by

mutation

pressure

increasing

the

AU

content

The

reason

UAG

is

reassigned

less

frequently

than

UGA

may

be

because

of

the

relative

difficulty

of

the

required

change

in

the

tRNA

In

the

case

of

UGA

the

existing

tRNA-Trp

can

simply

mutate

its

anticodon

In

the

UAG

case

it

is

necessary

to

create

a

new

tRNA-Leu

with

a

CUA

anticodon

to

pair

with

UAG

This

can

be

done

by

making

a

mutation

in

the

second

position

of

a

tRNA-Leu

with

a

CAA

anticodon

Such

a

mutation

in

the

anticodon

would

not

change

the

amino

acid

specificity

of

the

tRNA

because

the

anticodon

of

the

tRNA-Leu

does

not

act

as

an

identity

element

for

recognition

by

the

leucyl-tRNA

synthetase

-LRB-

Asahara

et

al.

-RRB-

in

contrast

to

most

other

amino

acyl-tRNA

synthetases

which

recognize

the

anticodon

However

this

can

only

be

done

after

duplication

of

the

tRNA-Leu

because

one

copy

of

this

gene

is

still

required

to

translate

UUG

Leu

codons

Thus

this

reassignment

is

relatively

rare

because

it

requires

a

prior

tRNA

duplication

It

is

interesting

to

note

that

in

most

genomes

both

UUA

and

UUG

would

be

Leu

and

there

would

be

a

single

tRNA

with

UAA

anticodon

to

translate

both

these

Another

peculiarity

of

Scenedesmus

is

that

UUA

codons

are

not

used

-LRB-

Kück

et

al.

-RRB-

but

UUG

codons

are

frequent

This

is

clearly

related

to

the

fact

that

the

tRNA-Leu

has

CAA

anticodon

in

this

species

UAG

has

been

reassigned

to

Ala

in

a

group

of

green

algae

that

are

closely

related

to

Scenedesmus

-LRB-

Hayashi-Ishimaru

et

al.

-RRB-

Complete

genomes

are

not

available

for

this

group

therefore

they

are

not

included

in

our

Fig.

It

seems

likely

that

UAG

disappeared

in

the

ancestor

of

Scenedesmus

and

the

other

algae

and

that

the

codon

was

captured

by

Leu

in

Scenedesmus

and

by

Ala

in

the

other

species

Table

also

shows

the

probability

of

disappearance

of

the

UAG

codon

for

these

two

cases

of

UAG

reassignment

This

is

calculated

in

the

same

way

as

for

UGA

above

These

values

do

not

appear

unreasonably

small

in

view

of

the

uncertainty

in

the

genome

properties

at

the

point

of

reassignment

Therefore

we

conclude

that

these

changes

are

consistent

with

the

CD

mechanism

Sense

Codon

Reassignments

Linked

to

Codon

Disappearance

It

is

not

only

Stop

codons

that

can

be

reassigned

by

the

CD

mechanism

The

following

examples

show

sense

codon

changes

occurring

via

this

mechanism

In

the

canonical

code

both

CUN

and

UUR

code

for

Leu

and

these

two

codon

blocks

are

accessible

to

one

another

by

a

synonymous

mutation

at

the

first

position

Similarly

CGN

and

AGR

both

code

for

Arg

and

are

also

accessible

to

one

another

via

a

synonymous

mutation

at

first

position

In

AU-rich

genomes

CUN

and

CGN

codon

families

are

subject

to

disappearance

and

replacement

by

UUR

and

AGR

Table

gives

the

number

of

occurrences

of

the

codon

groups

for

Leu

and

Arg

in

several

Fungi

Base

frequencies

at

FFD

sites

are

also

shown

C

and

G

frequencies

are

very

low

in

all

the

species

in

Table

indicating

a

strong

mutation

pressure

toward

A

and

U.

Table

Codon

usage

in

some

Fungi

lineages

intronic

ORFs

excluded

-LRB-

E

-RRB-

or

included

-LRB-

I

-RRB-

Leu

codonsArg

codonsIle

and

Met

CodonsFrequency

at

FFD

sitesCUNUURCGNAGRAUUAUCAUAAUG

U

C

A

GS

japonicus

-LRB-

E

-RRB-

S.

octosporus

-LRB-

E

-RRB-

S.

pombe

-LRB-

E

-RRB-

Y.

lipolytica

-LRB-

E

-RRB-

C.

stellata

-LRB-

E

-RRB-

C.

C.

parapsilosis

-LRB-

E

-RRB-

C.

parapsilosis

-LRB-

I

-RRB-

P.

canadensis

-LRB-

E

-RRB-

P.

canadensis

-LRB-

I

-RRB-

A.

K.

lactis

-LRB-

E

-RRB-

K.

lactis

-LRB-

I

-RRB-

K.

thermotolerans

-LRB-

E

-RRB-

K.

thermotolerans

-LRB-

I

-RRB-

C.

glabrata

-LRB-

E

-RRB-

C.

glabrata

-LRB-

I

-RRB-

S.

cerevisiae

-LRB-

E

-RRB-

S.

castellii

-LRB-

E

-RRB-

S.

servazzii

-LRB-

E

-RRB-

aCUN

is

reassigned

to

Thr

and

an

unusual

tRNA-Thr

gene

is

present

in

these

species.bThere

is

no

tRNA

for

the

CUN

block

in

this

species.cThe

tRNA-Arg

is

deleted

in

these

species.dThe

tRNA-Ile

-LRB-

CAU

-RRB-

is

deleted

in

these

species.eAUA

is

reassigned

to

Met

in

these

species

The

species

in

the

top

half

of

Table

-LRB-

P.

canadensis

and

above

-RRB-

use

CUN

for

Leu

as

in

the

canonical

code

These

species

possess

a

tRNA-Leu

with

anticodon

UAG

for

this

family

In

all

these

cases

the

CUN

family

is

less

frequent

than

the

UUR

family

The

six

species

followed

by

a

superscript

a

in

Table

have

undergone

a

reassignment

of

the

CUN

family

to

Thr

These

species

possess

an

unusual

tRNA-Thr

with

anticodon

UAG

-LRB-

Sibler

et

al.

Osawa

et

al.

-RRB-

and

the

usual

tRNA-Leu

-LRB-

UAG

-RRB-

is

not

found

in

the

genome

In

K.

lactis

the

codon

family

is

not

used

at

all

Significantly

this

is

the

only

species

in

which

no

tRNA

exists

that

pairs

with

this

codon

family

The

fact

that

the

tRNA-Thr

-LRB-

UAG

-RRB-

appears

only

in

species

where

the

tRNA-Leu

-LRB-

UAG

-RRB-

is

absent

suggests

that

the

new

gene

evolved

directly

from

the

old

tRNA-Leu

-LRB-

UAG

-RRB-

gene

This

requires

no

change

in

the

anticodon

but

changes

elsewhere

are

required

in

order

that

the

tRNA

is

recognized

by

the

threonyl-tRNA

synthetase

and

not

the

leucyl-tRNA

synthase

Although

many

amino

acyl-tRNA

synthetases

recognize

the

anticodon

recognition

of

tRNA-Leu

by

leucyl

tRNA-synthetase

is

exclusively

determined

by

the

bases

in

the

large

variable

arm

-LRB-

Asahara

et.al

-RRB-

Changes

in

the

variable

arm

could

have

prevented

the

tRNA

acting

as

a

tRNA-Leu

and

would

have

left

it

open

to

evolving

a

new

function

as

a

tRNA-Thr

The

codon

usage

figures

in

Table

indicate

that

the

change

is

attributable

to

the

CD

mechanism

The

number

of

occurrences

of

CUN

was

most

likely

driven

to

zero

prior

to

the

branching

of

A.

gossypii

-LRB-

see

Fig.

-RRB-

A

major

change

in

the

tRNA-Leu

-LRB-

UAG

-RRB-

gene

was

then

possible

allowing

it

to

be

charged

with

Thr

CUN

codons

later

reappeared

with

a

new

meaning

In

the

context

of

our

gain-loss

framework

-LRB-

Sengupta

and

Higgs

-RRB-

this

example

is

interesting

in

that

the

gain

and

loss

of

function

occur

in

the

same

tRNA

Although

it

is

likely

that

the

unusual

tRNA-Thr

in

these

species

evolved

directly

from

the

old

tRNA-Leu

the

alternative

scenario

is

that

there

was

a

duplication

of

the

tRNA-Thr

-LRB-

UGU

-RRB-

gene

that

decodes

the

normal

ACN

Thr

codons

One

of

these

genes

might

then

have

changed

its

anticodon

to

UAG

-LRB-

two

mutations

required

-RRB-

In

order

to

determine

the

origin

of

the

tRNA-Thr

-LRB-

UAG

-RRB-

gene

we

constructed

a

phylogeny

of

all

tRNAs

from

P.

canadensis

K.

thermotolerans

and

S.

castellii

i.e.

from

representative

species

before

and

after

the

codon

reassignment

The

tRNAs

of

each

type

formed

monophyletic

triplets

with

the

exception

of

the

genes

with

UAG

anticodons

The

tRNA-Thr

-LRB-

UAG

-RRB-

genes

in

K.

thermotolerans

and

S.

castellii

showed

no

close

relationship

to

either

the

tRNA-Thr

-LRB-

UGU

-RRB-

genes

or

the

tRNA-Leu

-LRB-

UAG

-RRB-

gene

in

P.

canadensis

Thus

neither

of

the

two

possible

scenarios

was

supported

Despite

this

it

stills

seems

most

likely

that

the

new

tRNA-Thr

gene

evolved

from

the

old

tRNA-Leu

a

conclusion

also

reached

by

Sibler

et

al.

-LRB-

-RRB-

and

Osawa

et

al.

-LRB-

-RRB-

One

possibility

that

can

not

entirely

be

ruled

out

from

the

codon

usage

data

is

that

the

changes

in

the

tRNA-Leu

-LRB-

UAG

-RRB-

occurred

when

the

CUN

codons

were

very

rare

but

not

entirely

absent

The

changes

might

have

been

such

as

to

immediately

cause

the

tRNA

to

be

charged

by

Thr

or

might

have

happened

more

gradually

so

that

the

same

tRNA

could

be

ambiguously

charged

by

Leu

and

Thr

during

the

changeover

period

We

would

then

have

to

count

this

as

an

example

of

the

AI

mechanism

Nevertheless

the

low

CUN

number

resulting

from

mutation

pressure

is

clearly

a

major

factor

in

this

case

and

we

therefore

feel

comfortable

in

classifying

it

under

the

CD

mechanism

The

tRNA

gene

has

undergone

considerable

modification

including

an

unusual

insertion

that

makes

the

anticodon

loop

larger

than

the

standard

seven

bases

In

our

opinion

it

is

unlikely

that

such

a

large

change

could

have

happened

while

the

tRNA

remained

simultaneously

functional

for

both

amino

acids

Table

also

shows

disappearances

in

the

Arg

codons

that

happen

in

parallel

with

those

in

the

Leu

codons

Mutation

pressure

away

from

C

causes

replacement

of

CGN

codons

by

AGR

Once

CGN

codons

have

disappeared

there

is

no

penalty

to

the

deletion

of

the

tRNA-Arg

-LRB-

ACG

-RRB-

gene

This

gene

is

absent

in

the

species

followed

by

a

superscript

c

in

Table

and

CGN

is

absent

in

these

species

-LRB-

or

apparently

there

is

just

one

in

C.

glabrata

-RRB-

On

the

other

hand

the

gene

is

still

present

in

S.

cerevisiae

and

K.

thermotolerans

and

a

handful

of

CGN

codons

still

remain

in

these

species

The

tRNA-Arg

-LRB-

ACG

-RRB-

genes

in

S.

cerevisiae

and

K.

thermotolerans

show

clear

sequence

homology

to

that

in

P.

canadensis

From

the

phylogeny

of

these

species

in

Fig.

we

see

that

it

requires

four

independent

deletions

of

the

tRNA-Arg

-LRB-

ACG

-RRB-

to

explain

the

observed

pattern

of

absences

of

the

gene

indicating

that

deletion

of

redundant

genes

is

a

rapid

phenomenon

Figure

shows

several

separate

reassignments

of

CGN

from

Arg

to

unassigned

and

only

one

reassignment

of

CUN

from

Leu

to

Thr

This

is

because

the

symbols

label

points

where

the

tRNAs

were

deleted

or

mutated

not

points

where

the

codon

disappeared

Our

interpretation

is

that

both

CUN

and

CGN

codons

were

absent

in

the

ancestor

of

the

group

of

yeast

species

prior

to

the

branching

of

A.

gossypii

and

that

change

in

the

tRNA-Leu

occurred

at

this

point

whereas

the

deletions

of

the

tRNA-Arg

genes

did

not

occur

till

after

the

split

between

the

species

This

would

permit

small

numbers

of

CGN

codons

to

reappear

in

some

lineages

where

the

gene

was

not

deleted

-LRB-

e.g.

S.

cerevisiae

and

K.

thermotolerans

-RRB-

The

final

twist

in

this

story

is

that

the

CUN

codons

are

absent

in

K.

lactis

This

means

that

there

must

have

been

a

further

reassignment

of

CUN

from

Thr

to

unassigned

in

this

species

only

Clearly

the

codons

have

disappeared

in

this

case

which

could

be

due

to

drift

or

because

of

poor

functioning

of

the

tRNA-Thr

-LRB-

UAG

-RRB-

gene

in

this

species

which

would

have

caused

selection

against

this

codon

block

We

have

little

evidence

in

this

case

but

for

the

purposes

of

our

summary

table

in

the

Discussion

below

-LRB-

Table

-RRB-

we

classified

it

as

CD

Table

shows

another

example

of

the

disappearance

of

the

CGN

block

in

Y.

lipolytica

The

corresponding

tRNA-Arg

is

also

deleted

in

this

species

-LRB-

Kerscher

et

al.

-RRB-

In

S.

pombe

and

C.

stellata

the

species

that

branch

before

and

after

Y.

lipolytica

the

tRNA-Arg

is

still

present

and

the

CGN

codons

are

still

used

CGN

has

thus

become

unassigned

in

this

single

species

This

also

appears

to

be

a

result

of

mutation

pressure

causing

disappearance

of

CGN

before

deletion

of

the

tRNA-Arg

The

Probability

of

Disappearance

of

CUN

and

CGN

Codons

The

number

of

Leu

and

Arg

codons

in

these

genomes

is

much

larger

than

the

number

of

stops

Therefore

it

is

more

difficult

for

these

sense

codons

to

disappear

than

it

is

for

stops

Nevertheless

the

probability

of

CD

is

not

unreasonably

low

in

these

cases

If

we

assume

that

all

six

Leu

codons

are

in

equilibrium

we

find

from

which

the

sum

of

the

relative

frequencies

of

all

the

CUN

block

codons

is

The

probability

of

disappearance

of

the

CUN

block

is

where

NLeu

is

the

number

of

Leu

codons

in

the

genome

However

Table

shows

that

this

calculation

is

hardly

necessary

In

A.

gossypii

the

species

branching

immediately

after

the

proposed

point

of

the

codon

reassignment

C

at

FFD

sites

i.e.

the

mutational

bias

against

C

is

so

strong

that

there

are

no

C

bases

at

all

in

any

FFD

site

Thus

fCUN

and

Pdis

Presumably

the

chance

of

mutating

to

a

C

is

not

precisely

zero

in

A.

gossypii

but

it

is

small

enough

that

all

the

FFD

C-ending

codons

disappear

Therefore

it

is

not

unreasonable

that

the

CUN

block

should

have

disappeared

in

the

ancestor

of

this

group

As

we

do

not

know

that

C

was

exactly

zero

in

the

ancestor

we

would

like

to

know

how

small

it

must

have

been

in

order

for

there

to

be

a

significant

probability

of

disappearance

of

CUN

It

can

be

seen

that

C

is

low

in

all

species

in

Table

but

variations

of

make

a

big

difference

in

estimation

of

Pdis

Estimation

of

base

frequencies

is

further

complicated

by

the

presence

of

intronic

ORFs

in

many

of

these

species

It

has

previously

been

shown

that

codon

usage

patterns

differ

between

the

intronic

ORFs

and

the

other

genes

-LRB-

Bullerwell

et

al.

Talla

et

al.

-RRB-

For

several

species

in

Table

we

have

listed

separate

codon

information

for

the

genes

excluding

the

intronic

ORFs

-LRB-

E

-RRB-

and

for

the

full

set

of

genes

including

the

intronic

ORFs

-LRB-

I

-RRB-

In

every

case

considered

C

is

slightly

higher

if

the

intronic

ORFs

are

included

These

ORFs

vary

in

number

between

species

and

are

not

present

in

some

species

including

A.

gossypii

This

makes

it

difficult

to

know

the

number

of

genes

in

the

ancestral

genome

at

the

point

of

CUN

reassignment

It

also

suggests

that

these

sequences

may

be

recent

insertions

and

that

the

base

composition

may

not

have

adjusted

to

the

equilibrium

base

composition

of

the

rest

of

the

genome

Furthermore

the

presence

of

rare

codons

in

the

intronic

ORFs

suggests

that

these

sequences

may

not

be

expressed

at

a

high

level

or

that

their

expression

may

not

be

important

for

the

organism

For

example

in

K.

lactis

and

K.

thermotolerans

AUA

is

avoided

because

of

deletion

of

the

corresponding

tRNA

-LRB-

to

be

discussed

below

-RRB-

but

there

are

substantially

more

AUA

codons

in

the

intronic

ORFs

than

the

regular

genes

For

these

reasons

it

is

likely

that

the

base

frequencies

in

the

genes

excluding

the

intronic

ORFs

are

better

indicators

of

the

equilibrium

base

frequencies

of

the

mutational

process

affecting

the

most

important

genes

in

the

genome

We

also

note

that

the

presence

of

the

intronic

ORFs

is

sometimes

associated

with

alternative

splicing

so

that

the

same

exon

is

used

in

several

proteins

-LRB-

e.g.

S.

cerevisiae

and

C.

stellata

-RRB-

Clearly

we

do

not

want

to

count

the

same

exon

twice

when

counting

codons

A

full

investigation

of

all

these

effects

on

intronic

ORFs

is

beyond

the

scope

of

this

paper

Given

all

the

caveats

above

it

is

clear

that

any

estimate

of

Pdis

is

subject

to

a

very

large

uncertainty

However

we

wish

to

give

a

fair

estimate

for

a

case

where

C

is

not

exactly

zero

We

use

the

figures

for

C.

glabrata

because

the

base

frequencies

with

and

without

the

intronic

ORFs

differ

less

than

in

most

species

To

be

conservative

we

use

the

figures

including

the

ORFs

From

Table

we

obtain

fCUN

and

Pdis

-LRB-

-RRB-

Slight

reductions

in

C

would

increase

this

figure

by

orders

of

magnitude

We

conclude

the

bias

against

C

must

have

been

at

least

as

strong

as

it

is

in

C.

glabrata

-LRB-

i.e.

C

-RRB-

in

order

for

the

CUN

codons

to

have

a

reasonable

chance

of

disappearing

The

fact

that

C

becomes

exactly

zero

in

A.

gossypii

and

is

less

than

in

several

other

cases

in

Table

shows

that

these

extreme

biases

do

occur

Swire

et

al.

-LRB-

-RRB-

have

calculated

many

examples

of

CD

probabilities

The

CUN

reassignment

corresponds

to

case

of

Swire

et

al.

-LRB-

Table

-RRB-

For

some

reason

they

estimated

a

vanishingly

small

probability

of

disappearance

of

the

CUN

codons

-LRB-

Pdis

-RRB-

which

is

the

lowest

of

any

of

the

cases

they

list

They

thus

argued

that

CD

was

impossible

in

this

case

We

disagree

since

we

have

shown

that

Pdis

is

high

due

to

the

extreme

bias

against

C

bases

This

case

is

exceptional

however

In

all

the

other

examples

of

sense-to-sense

codon

reassignments

discussed

by

Swire

et

al.

we

agree

that

the

probability

of

CD

is

indeed

unrealistically

low

and

that

the

reassignment

must

have

occurred

without

CD

Using

an

argument

similar

to

that

above

the

relative

frequency

of

CGN

codons

is

and

the

probability

of

disappearance

of

the

CGN

block

is

We

have

already

seen

that

C

in

A.

gossypii

and

is

very

low

in

related

species

NArg

is

much

lower

than

NLeu

so

disappearance

of

CGN

in

the

ancestor

of

this

group

is

therefore

quite

likely

and

is

easier

than

disappearance

of

CUN

For

the

case

of

Y.

lipolytica

if

we

use

the

figures

from

this

species

as

a

proxy

for

the

ancestor

we

obtain

Pdis

-LRB-

-RRB-

which

is

not

unreasonably

small

Clearly

it

is

much

more

difficult

for

sense

codons

to

disappear

than

stop

codons

as

the

number

of

codons

involved

is

much

larger

For

this

reason

we

need

an

extremely

strong

base

frequency

bias

in

order

to

cause

disappearance

However

in

the

above

cases

we

have

shown

that

the

bias

is

indeed

extreme

and

that

the

CD

mechanism

seems

likely

A

reviewer

of

this

paper

pointed

out

that

Osawa

and

Jukes

-LRB-

-RRB-

the

original

proponents

of

the

CD

model

insisted

that

according

to

their

theory

every

single

occurrence

of

the

codon

must

disappear

from

the

genome

before

the

reassignment

occurred

From

current

genome

data

it

will

never

be

possible

to

tell

whether

every

occurrence

of

the

codon

disappeared

at

the

time

the

reassignment

occurred

Therefore

if

we

insist

on

this

strict

definition

it

is

impossible

to

assign

a

mechanism

to

any

of

the

reassignment

events

However

to

be

more

constructive

and

more

practical

we

wish

to

make

a

clear

distinction

between

the

examples

discussed

above

and

those

that

we

discuss

in

the

remainder

of

the

paper

In

the

above

examples

there

is

a

clear

case

that

CD

is

likely

due

to

the

direction

of

the

mutation

pressure

and

the

best

estimates

of

the

CD

probabilities

are

not

too

low

The

extreme

rarity

-LRB-

and

probably

absence

-RRB-

of

these

codons

was

clearly

a

major

factor

in

allowing

the

codon

reassignment

to

occur

In

contrast

in

the

examples

below

there

is

clear

evidence

that

the

codon

was

not

rare

at

the

time

of

the

reassignment

and

that

the

codon

reassignment

was

initiated

by

some

other

event

not

related

to

CD

Reassignments

That

Can

not

Be

Explained

by

Codon

Disappearance

Reassignment

of

AUA

from

Ile

to

Met

Is

Initiated

by

Loss

of

a

tRNA-Ile

Gene

In

the

canonical

code

the

AUN

codons

are

unusual

in

that

AUU

AUC

and

AUA

code

for

Ile

and

only

AUG

codes

for

Met

The

tRNAs

need

to

distinguish

between

codons

ending

in

A

and

those

ending

in

G

which

is

not

usually

necessary

-LRB-

except

for

the

UGG

Trp

codon

-RRB-

The

translation

system

in

bacteria

uses

two

tRNAs

for

Ile

one

with

anticodon

GAU

which

translates

AUY

codons

and

one

with

anticodon

CAU

which

translates

AUA

only

It

is

known

from

studies

on

E.

coli

-LRB-

Muramatsu

et

al.

-RRB-

that

the

C

at

the

wobble

position

undergoes

a

posttranscriptional

base

modification

to

-LRB-

lysidine

-RRB-

which

allows

this

tRNA-Ile

to

pair

with

the

AUA

codon

The

same

gene

also

is

present

in

many

mitochondrial

genomes

in

which

AUA

is

translated

as

Ile

and

it

is

presumed

that

the

same

base

modification

occurs

However

the

tRNA-Ile

-LRB-

CAU

-RRB-

gene

is

absent

in

mitochondrial

genomes

in

which

AUA

is

reassigned

to

Met

Before

codon

reassignment

tRNA-Met

also

has

a

CAU

anticodon

which

pairs

with

AUG

only

but

modification

of

the

wobble-position

C

allows

it

to

pair

with

both

AUA

and

AUG

-LRB-

Tomita

et

al.

-RRB-

In

order

to

locate

and

understand

the

cases

of

AUA

codon

reassignment

in

mitochondria

it

is

crucial

to

establish

which

genomes

contain

the

tRNA-Ile

-LRB-

CAU

-RRB-

gene

This

is

complicated

by

the

fact

that

some

genomes

contain

three

tRNAs

with

CAU

anticodons

-LRB-

a

Met

initiator

a

Met

elongator

and

an

Ile

-RRB-

and

the

Ile

tRNA

is

often

mistakenly

annotated

as

Met

To

uncover

misannotations

we

constructed

a

phylogeny

of

all

the

tRNAs

with

CAU

anticodon

from

all

the

fungi

genomes

in

our

data

set

The

genes

fell

into

three

groups

that

could

be

reliably

identified

Supplementary

Table

lists

the

position

of

the

three

tRNAs

in

each

genome

and

hence

shows

in

which

genomes

there

have

been

gene

deletions

From

this

we

note

that

the

tRNA-Ile

is

missing

from

the

seven

species

from

A.

gossypii

downward

in

Table

Hence

this

gene

must

have

been

deleted

prior

to

the

branching

of

A.

gossypii

as

shown

in

Fig.

Before

the

deletion

of

the

tRNA-Ile

-LRB-

CAU

-RRB-

gene

AUA

and

AUU

are

frequent

codons

for

Ile

but

AUC

is

rare

-LRB-

e.g.

C.

albicans

C.

parapsiplopsis

and

P.

canadensis

-RRB-

This

is

to

be

expected

from

the

FFD

base

frequencies

Mutation

pressure

is

therefore

in

the

wrong

direction

to

cause

disappearance

of

AUA

and

in

fact

AUA

is

the

most

frequent

Ile

codon

in

P.

canadensis

the

closest

relative

to

the

species

concerned

We

therefore

argue

that

this

reassignment

was

initiated

by

loss

of

the

tRNA-Ile

while

the

AUA

codon

was

frequent

in

the

genome

Hence

it

is

an

example

of

the

UC

mechanism

according

to

our

classification

Immediately

after

the

loss

of

this

tRNA

the

AUA

codon

had

no

specific

tRNA

to

pair

with

it

and

it

may

be

said

to

have

been

unassigned

The

tRNA

loss

could

not

have

been

lethal

however

so

there

must

have

been

another

way

of

decoding

AUAs

In

Metazoa

some

genomes

are

known

where

the

tRNA-Ile

-LRB-

CAU

-RRB-

gene

has

been

deleted

but

the

AUA

codon

has

not

been

reassigned

Yokobori

et

al.

-LRB-

-RRB-

have

shown

that

in

such

cases

AUA

is

translated

as

Ile

by

the

tRNA-Ile

-LRB-

GAU

-RRB-

even

though

this

requires

GA

mispairing

at

the

wobble

position

It

is

presumed

that

this

is

tolerated

if

there

is

no

better

alternative

available

i.e.

if

the

tRNA-Ile

-LRB-

CAU

-RRB-

has

been

deleted

but

no

modification

to

the

tRNA-Met

has

occurred

This

may

be

what

is

happening

in

the

two

Kluyveromyces

species

where

usage

of

AUA

is

very

low

despite

the

fact

that

A

is

also

frequent

at

FFD

sites

in

these

species

This

demonstrates

a

definite

avoidance

of

AUA

probably

due

to

the

fact

that

there

is

no

tRNA

that

translates

the

AUA

codon

efficiently

Talla

et

al.

-LRB-

-RRB-

argue

that

AUA

is

assigned

to

Met

in

K.

thermotolerans

based

on

only

two

occurrences

of

this

codon

in

the

gene

It

may

be

more

reasonable

to

say

that

AUA

is

unassigned

in

both

K.

lactis

and

K.

thermotolerans

In

C.

glabrata

Koszul

et

al.

-LRB-

-RRB-

conclude

that

AUA

is

associated

with

Met

based

on

comparison

of

C.

glabrata

sequences

with

S.

cerevisiae

sequences

from

which

they

find

that

of

AUA

codons

in

C.

glabrata

appear

at

conserved

Met

sites

encoded

by

AUA

in

S.

cerevisiae

There

is

still

some

avoidance

of

AUA

codons

in

C.

glabrata

nevertheless

In

A.

gossypii

and

the

Saccharomyces

species

AUA

is

frequent

and

we

presume

that

it

is

efficiently

translated

as

Met

This

may

imply

that

there

have

been

two

independent

adaptations

of

the

tRNA-Met

to

deal

with

AUA

in

these

two

lineages

We

wish

to

underline

the

difference

between

the

AUA

reassignment

and

the

reassignments

of

CUN

and

CGN

in

the

same

species

-LRB-

see

previous

section

-RRB-

For

CUN

and

CGN

mutation

pressure

drives

the

disappearance

In

both

these

cases

there

is

only

one

tRNA

for

the

block

of

four

codons

and

if

this

were

deleted

-LRB-

in

the

CGN

case

-RRB-

or

drastically

mutated

-LRB-

as

probably

happened

in

the

CUN

case

-RRB-

there

would

be

no

plausible

alternative

tRNA

that

could

step

in

Hence

these

changes

can

only

occur

after

the

codon

disappears

In

contrast

mutation

pressure

leads

to

frequent

usage

of

AUA

for

Ile

Loss

of

the

tRNA-Ile

-LRB-

CAU

-RRB-

is

possible

without

CD

because

the

tRNA-Ile

-LRB-

GAU

-RRB-

and

possibly

also

the

tRNA-Met

-LRB-

CAU

-RRB-

has

some

ability

to

pair

with

this

codon

A

calculation

of

the

AUA

CD

probability

also

serves

to

highlight

the

difference

with

the

previous

section

If

the

three

Ile

codons

are

in

equilibrium

and

Using

C.

glabrata

-LRB-

I

-RRB-

as

an

example

as

we

did

for

the

CUN

disappearance

we

obtain

Pdis

-LRB-

-RRB-

which

is

indeed

vanishingly

low

As

described

in

the

previous

paragraph

the

adaptation

to

using

AUA

for

Met

seems

to

have

been

gradual

A

possible

reason

for

this

could

be

that

the

change

in

the

tRNA-Met

is

due

to

a

base

modification

of

the

wobble

position

C

not

a

mutation

-LRB-

as

in

the

tRNA-Trp

where

the

wobble

position

C

mutates

to

U

when

the

UGA

codon

is

reassigned

to

Trp

-RRB-

The

difference

between

the

avoidance

of

AUA

seen

in

Kluyveromyces

and

the

fairly

frequent

use

of

AUA

as

Met

in

Saccharomyces

may

lie

in

the

fraction

of

tRNAs

in

which

this

base

gets

modified

The

other

species

of

fungus

in

which

tRNA-Ile

-LRB-

CAU

-RRB-

is

missing

is

Schizosaccharomyces

octosporus

although

the

gene

is

still

present

in

S.

pombe

and

S.

japonicus

Table

shows

that

AUA

is

absent

altogether

in

S.

octosporus

-LRB-

excluding

intronic

ORFs

-RRB-

whereas

comparison

with

the

other

two

species

suggests

that

there

were

or

AUA

codons

present

before

the

loss

of

the

tRNA

Once

again

it

is

unlikely

that

these

codons

would

have

disappeared

by

chance

and

A

is

a

frequent

base

at

FFD

sites

in

all

three

Schizosaccharomyces

species

Hence

the

disappearance

of

AUA

in

S.

octosporus

is

due

to

selection

against

this

codon

after

the

loss

of

the

tRNA

Bullerwell

et

al.

-LRB-

-RRB-

show

that

AUA

has

not

disappeared

entirely

in

the

intronic

ORFs

of

S.

octosporus

This

suggests

that

there

is

weaker

selection

on

codon

usage

in

the

intronic

ORFs

because

these

genes

are

less

strongly

expressed

than

the

standard

genes

It

also

confirms

that

selection

must

have

driven

the

disappearance

of

AUA

in

the

standard

genes

because

if

mutation

pressure

were

responsible

it

would

have

affected

the

intronic

ORFs

equally

Thus

we

conclude

that

the

initial

event

must

have

been

the

deletion

of

the

tRNA-Ile

-LRB-

CAU

-RRB-

and

hence

this

corresponds

to

the

UC

mechanism

In

this

case

the

codon

has

not

been

reassigned

to

Met

but

there

is

the

potential

for

this

to

occur

in

the

future

if

there

is

a

gain

of

function

in

the

tRNA-Met

From

Supplementary

Table

we

see

that

C.

albicans

C.

parapsilosis

and

Y.

lipolytica

have

only

one

tRNA-Met

Possibly

this

plays

the

role

of

both

initiator

and

elongator

We

do

not

consider

it

further

because

this

deletion

is

not

associated

with

any

codon

reassignment

AUA

is

also

reassigned

to

Met

in

some

Metazoa

The

tRNA-Ile

-LRB-

CAU

-RRB-

gene

is

present

in

close

relatives

of

the

Metazoa

Amoebidium

and

Monosiga

-LRB-

Burger

et

al.

-RRB-

and

also

in

Porifera

-LRB-

Lavrov

et

al.

-RRB-

AUA

is

Ile

in

these

species

The

gene

is

deleted

in

all

remaining

Metazoa

-LRB-

see

Fig.

-RRB-

which

suggests

that

it

was

lost

after

the

divergence

of

the

phylum

Porifera

from

other

metazoan

phyla

Cnidaria

are

unusual

in

that

they

have

lost

almost

all

tRNAs

from

the

mitochondrial

genome

and

require

import

of

tRNAs

from

the

nucleus

AUA

remains

Ile

in

this

group

presumably

because

the

tRNA-Ile

-LRB-

CAU

-RRB-

is

imported

There

are

also

two

other

groups

in

which

AUA

remains

as

Ile

Platyhelminthes

or

more

specifically

Rhabditophora

-LRB-

Telford

et

al.

-RRB-

and

Echinoderms/Hemichordates

These

phyla

retain

sufficient

tRNAs

to

translate

the

full

code

and

there

is

no

suggestion

of

tRNA

import

It

therefore

appears

that

AUA

is

translated

as

Ile

by

the

tRNA-Ile

-LRB-

GAU

-RRB-

This

is

consistent

with

the

argument

of

Yokobori

et

al.

-LRB-

-RRB-

that

GA

pairing

at

the

anticodon

wobble

position

is

tolerated

if

there

is

no

better

alternative

anticodon-codon

pairing

solution

available

In

the

remaining

metazoan

phyla

AUA

is

reassigned

to

Met

The

usual

tRNA-Met

anticodon

is

CAU

This

can

be

modified

in

several

different

ways

to

pair

with

AUA

in

addition

to

AUG.

In

Drosophila

modification

from

C

to

-LRB-

-RRB-

at

the

wobble

position

occurs

-LRB-

Tomita

et

al.

-RRB-

These

authors

also

show

that

the

unmodified

CAU

anticodon

can

translate

both

codons

if

the

base

in

the

position

of

the

tRNA

is

-LRB-

-RRB-

instead

of

A

-LRB-

see

also

Kuchino

et

al.

-RRB-

Note

that

the

position

is

the

nucleotide

immediately

of

the

anticodon

In

urochordates

the

tRNA-Met

has

a

U

AU

anticodon

-LRB-

where

U

is

an

unknown

derivative

of

U

-RRB-

This

is

a

third

means

by

which

the

tRNA-Met

has

gained

the

ability

to

translate

AUA

There

is

insufficient

evidence

to

say

exactly

where

these

modifications

to

the

tRNA-Met

evolved

and

there

are

many

species

for

which

the

details

of

the

tRNA-Met

are

not

known

Nevertheless

the

existence

of

these

different

modifications

to

the

tRNA-Met

suggests

that

the

reassignment

of

AUA

to

Met

has

occurred

independently

at

least

three

times

in

different

phyla

after

the

loss

of

the

original

tRNA-Ile

-LRB-

CAU

-RRB-

gene

-LRB-

as

shown

in

Fig.

-RRB-

Note

that

the

AUA

codon

is

common

in

Porifera

and

Cnidaria

and

we

did

not

find

any

example

of

disappearance

of

this

codon

in

any

of

the

Metazoa

-LRB-

see

Table

-RRB-

Hence

there

is

no

suggestion

that

the

codon

disappeared

before

the

loss

of

the

tRNA-Ile

-LRB-

CAU

-RRB-

As

the

tRNA

loss

occurred

first

this

is

an

example

of

the

UC

mechanism

very

similar

to

the

case

of

AUA

reassignment

in

Fungi

Table

Codon

usage

in

some

metazoan

lineagesIle

and

Met

codonsSer

and

Arg

codonsAsn

and

Lys

codonsFrequency

at

FFD

sitesAUUAUCAUAAUGAGUAGCAGAAGGAAUAACAAAAAG

U

C

A

GAxinella

Geodia

Metridium

Acropora

Limulus

Daphnia

Drosophila

Caenorhabditis

Trichinella

Katharina

Lumbricus

Terebratulina

Fasciola

Schistosoma

Taenia

Paracentrotus

Asterina

Balanoglossus

Saccoglossus

Halocynthia

Cionia

Branchiostoma

Branchiostoma

Epigonichthys

Myxine

Homo

aAUA

is

Met

in

these

species.bAGR

remains

Arg.cAGR

is

Gly.dAGR

is

Stop.eAGG

is

avoided

probably

because

there

is

an

unmodified

G

in

the

wobble

position

of

tRNA-Ser

-LRB-

GCU

-RRB-

fAAA

is

reassigned

to

Asn

We

interpret

the

decoding

of

AUA

as

Ile

in

Platyhelminths

and

Echinoderms/Hemichordates

as

indicating

that

an

appropriate

change

in

the

tRNA-Met

did

not

occur

and

that

AUA

has

always

been

Ile

in

these

lineages

An

alternative

is

that

AUA

was

assigned

to

Met

once

at

the

point

of

the

loss

of

the

tRNA-Ile

-LRB-

CAU

-RRB-

and

that

there

were

reversals

of

the

change

in

these

two

groups

-LRB-

as

shown

in

Fig.

of

Knight

et

al.

-LSB-

-RSB-

and

Fig.

of

Yokobori

et

al.

-LSB-

-RSB-

-RRB-

However

this

scenario

does

not

explain

why

more

than

one

type

of

modification

of

the

tRNA-Met

should

exist

Also

once

the

AUA

codon

is

captured

by

Met

and

mutations

have

occurred

throughout

the

genome

-LRB-

so

that

AUA

appears

in

positions

where

a

Met

rather

than

an

Ile

is

required

-RRB-

then

the

organism

is

better

adapted

than

it

would

be

if

AUA

were

inefficiently

translated

by

the

tRNA-Ile

-LRB-

GAU

-RRB-

Thus

we

see

no

reason

why

a

reversal

of

this

change

should

occur

Telford

et

al.

-LRB-

-RRB-

have

also

discussed

these

two

alternative

scenarios

in

a

parsimony

treatment

however

they

did

not

consider

the

position

of

the

tRNA-Ile

-LRB-

CAU

-RRB-

gene

loss

or

the

fact

that

there

are

several

independent

modifications

to

the

tRNA-Met

both

of

which

are

essential

points

in

our

interpretation

Reassignment

of

AUA

from

Ile

to

Met

has

also

been

observed

-LRB-

Ehara

et

al.

-RRB-

in

some

species

of

yellow-green

algae

-LRB-

Xanthophytes

-RRB-

The

entire

genome

of

these

species

is

not

yet

available

and

the

lack

of

information

about

the

tRNA

genes

and

codon

usage

makes

it

impossible

to

deduce

the

mechanism

of

change

However

it

is

clear

that

the

change

occurred

independently

of

similar

changes

observed

in

Metazoa

and

Fungi

Reassignments

Involving

the

AGR

Block

Are

Initiated

by

Loss

of

a

tRNA-Arg

Gene

The

AGR

block

assigned

to

Arg

in

the

canonical

code

has

undergone

multiple

reassignments

in

Metazoa

The

tRNA-Arg

-LRB-

UCU

-RRB-

is

present

in

Porifera

but

is

absent

in

all

other

Metazoa

In

Cnidaria

almost

all

tRNAs

are

missing

and

tRNA

import

from

the

cytoplasm

is

required

This

is

not

associated

with

codon

reassignment

In

the

remaining

Metazoa

-LRB-

i.e.

Bilateria

-RRB-

the

loss

of

the

tRNA-Arg

-LRB-

UCU

-RRB-

gives

rise

to

subsequent

changes

in

the

genetic

code

Loss

of

this

gene

leaves

the

tRNA-Ser

-LRB-

GCU

-RRB-

as

the

only

candidate

for

translating

these

codons

Hence

AGR

is

reassigned

to

Ser

at

the

base

of

the

Bilateria

-LRB-

Fig.

-RRB-

This

is

classed

as

the

UC

mechanism

as

it

is

initiated

by

a

tRNA

loss

Note

that

AGR

codons

are

frequent

in

Porifera

and

Cnidaria

therefore

there

is

no

suggestion

of

CD

-LRB-

Table

-RRB-

The

GCU

anticodon

of

tRNA-Ser

usually

interacts

with

only

AGY

codons

prior

to

the

loss

of

the

tRNA-Arg

However

pairing

of

the

tRNA-Ser

with

AGR

codons

is

also

possible

to

some

extent

once

there

is

no

longer

any

competition

from

the

tRNA-Arg

-LRB-

Yokobori

et

al.

Yokoyama

et

al.

-RRB-

In

Drosophila

it

is

known

that

the

tRNA-Ser

-LRB-

GCU

-RRB-

translates

AGA

-LRB-

Tomita

et

al.

-RRB-

i.e.

there

is

a

GA

mispairing

at

the

wobble

position

in

the

same

way

as

in

the

mispairing

of

AUA

with

tRNA-Ile

-LRB-

GAU

-RRB-

after

the

loss

of

the

tRNA-Ile

-LRB-

CAU

-RRB-

In

Drosophila

AGG

is

absent

which

may

indicate

that

this

codon

is

avoided

because

of

selection

against

an

unfavorable

GG

mismatch

that

would

occur

at

the

wobble

position

It

should

also

be

noted

that

G

and

C

are

very

low

in

Drosophila

-LRB-

Table

-RRB-

so

the

absence

of

AGG

may

simply

be

a

result

of

mutation

pressure

In

Daphnia

there

is

only

one

AGG

codon

even

though

G

is

high

This

suggests

definite

avoidance

of

this

codon

However

not

all

arthropods

show

this

pattern

As

an

example

there

are

AGG

codons

remaining

in

Limulus

Two

other

groups

in

which

AGG

is

absent

or

very

rare

are

the

hemichordates

and

cephalochordates

Here

the

tRNA

also

has

a

G

at

the

wobble

position

which

we

presume

is

unmodified

In

contrast

Table

shows

many

other

cases

where

AGA

and

AGG

are

both

frequent

In

several

invertebrate

groups

the

G

wobble

position

is

mutated

to

a

U

which

allows

it

to

pair

with

all

the

AGN

codons

This

is

the

case

with

C.

elegans

and

T.

spiralis

in

Table

where

we

see

relatively

high

usage

of

AGG

Alternatively

in

some

echinoderms

-LRB-

Matsuyama

et

al.

-RRB-

and

mollusks

-LRB-

Tomita

et

al.

-RRB-

a

base

modification

from

G

to

also

permits

it

to

decode

the

entire

AGN

block

as

Ser

The

AGR

block

has

undergone

further

changes

in

urochordates

where

it

is

reassigned

to

Gly

and

craniates

where

it

is

reassigned

to

Stop

The

most

likely

state

prior

to

these

changes

would

be

that

AGA

was

moderately

frequent

and

AGG

was

rare

or

absent

-LRB-

as

with

the

current

hemichordates

and

cephalochordates

-RRB-

Urochordates

contain

an

additional

tRNA-Gly

with

anticodon

UCU

-LRB-

Gissi

et

al.

-RRB-

This

has

arisen

by

duplication

of

the

standard

tRNA-Gly

-LRB-

UCC

-RRB-

followed

by

anticodon

mutation

This

new

tRNA

pairs

efficiently

with

AGR

codons

and

outcompetes

the

tRNA-Ser

-LRB-

GCU

-RRB-

AGR

codons

become

very

frequent

after

the

reassignment

-LRB-

Table

-RRB-

because

mutation

from

GGR

glycine

codons

occurs

Note

that

A

is

greater

than

G

in

all

these

genomes

so

mutation

pressure

favors

synonymous

mutations

from

GGR

to

AGR

Since

AGA

was

a

moderately

frequent

codon

for

Ser

prior

to

the

reassignment

and

since

A

is

high

we

can

not

argue

that

mutation

pressure

caused

the

disappearance

of

AGA

and

replacement

by

AGR

prior

to

the

change

Thus

it

seems

unlikely

that

CD

is

responsible

for

this

change

This

argument

also

applies

for

the

reassignment

of

AGR

to

Stop

in

the

craniates

AGA

is

rare

in

craniates

after

the

change

because

Stop

codons

are

always

rare

These

genomes

have

very

high

A

so

unless

there

was

a

short

period

when

mutation

pressure

was

in

the

opposite

direction

to

that

seen

in

all

the

current

genomes

then

we

must

suppose

that

this

reassignment

occurred

without

disappearance

of

the

codon

This

is

puzzling

since

it

implies

premature

termination

of

translation

of

genes

containing

AGA

Ser

codons

Spruyt

et

al.

-LRB-

-RRB-

argued

that

AGA

is

also

read

as

Gly

in

Branchiostoma

lanceolatum

They

found

a

putative

tRNA

with

anticodon

UCU

that

may

have

arisen

by

duplication

and

mutation

of

the

tRNA-Gly

-LRB-

UCC

-RRB-

as

occurred

in

the

urochordates

However

the

codon

usage

numbers

suggest

that

this

is

not

the

case

and

that

the

sequence

identified

does

not

function

as

a

tRNA-Gly

The

pattern

seen

for

AGA

and

AGG

in

B.

lanceolatum

resembles

that

in

the

hemichordates

and

some

of

the

arthropods

where

there

is

a

single

tRNA-Ser

-LRB-

GCU

-RRB-

Also

other

cephalochordate

genomes

are

now

available

including

B.

floridae

-LRB-

Boore

et

al.

-RRB-

and

Epigonichthys

maldivensis

-LRB-

Nohara

et

al.

-RRB-

which

also

show

no

evidence

of

reassignment

of

AGA

to

Gly

The

distinction

between

B.

lanceolatum

and

B.

floridae

-LRB-

e.g.

Knight

et

al.

-RRB-

therefore

seems

unfounded

Reassignment

of

AAA

from

Lys

to

Asn

May

Proceed

Via

an

Ambiguous

Intermediate

The

reassignment

of

AAA

from

Lys

to

Asn

occurred

independently

in

echinoderms

and

platyhelminthes

Furthermore

the

AAA

codon

is

absent

in

the

hemichordate

Balanoglossus

-LRB-

see

Table

-RRB-

but

present

in

the

related

species

Saccoglossus

where

it

is

translated

as

Lys

as

in

the

canonical

code

-LRB-

M.J.

Smith

personal

communication

-RRB-

Most

metazoans

in

which

AAA

retains

its

standard

assignment

of

Lys

have

one

tRNA-Lys

-LRB-

UUU

-RRB-

which

pairs

with

AAR

codons

and

one

tRNA-Asn

-LRB-

GUU

-RRB-

which

pairs

with

AAY

codons

Mispairing

between

a

G

in

the

anticodon

wobble

position

and

an

A-ending

codon

is

implicated

in

both

the

reassignment

of

AUA

from

Ile

to

Met

and

the

reassignment

of

AGR

from

Arg

to

Ser

as

discussed

above

In

the

case

of

the

tRNA-Asn

-LRB-

GUU

-RRB-

mispairing

with

AAA

is

prevented

by

posttranscriptional

modification

of

the

wobble

position

G

to

queuosine

-LRB-

Q

-RRB-

which

allows

pairing

with

U

and

C

but

inhibits

pairing

with

A

-LRB-

Morris

et

al.

-RRB-

In

echinoderms

and

platyhelminthes

the

tRNA-Lys

anticodon

is

mutated

to

CUU

-LRB-

Tomita

et

al.

-RRB-

which

pairs

only

with

AAG

Several

changes

to

the

tRNA-Asn

occur

that

increase

its

ability

to

pair

with

AAA

The

modification

of

G

to

Q

does

not

take

place

-LRB-

Yokobori

et

al.

-RRB-

which

may

allow

some

degree

of

GA

mispairing

In

echinoderms

modification

of

the

second

anticodon

position

from

U

to

Psi

-LRB-

pseudouridine

-RRB-

enhances

the

ability

of

the

GPsiU-Asn

anticodon

to

recognize

the

AAA

codon

Also

in

some

echinoderms

the

mutation

of

the

base

immediately

adjacent

to

the

end

of

the

anticodon

from

a

U

to

a

C

seems

to

help

the

GUU

anticodon

to

recognize

AAA

-LRB-

Castrasena

et

al.

-RRB-

In

Balanoglossus

the

tRNA-Lys

has

also

changed

to

CUU

but

there

seem

to

be

no

modifications

in

the

tRNA-Asn

that

would

allow

AAA

to

be

reassigned

to

Asn

AAA

is

therefore

unassigned

In

all

groups

in

which

AAA

is

still

Lys

it

is

a

relatively

frequent

codon

and

it

is

usually

more

frequent

than

AAG

A

is

usually

quite

high

at

FFD

sites

Thus

even

though

Castrasena

et

al.

argue

for

the

CD

mechanism

it

seems

unlikely

to

us

that

AAA

disappeared

in

either

the

ancestor

of

the

platyhelminthes

or

the

echinoderms

Note

that

the

absence

of

AAA

in

Balanoglossus

is

not

shared

with

Saccoglossus

so

we

can

not

use

this

to

argue

that

AAA

disappeared

in

the

common

ancestor

of

echinoderms

and

hemichordates

The

AI

mechanism

seems

the

most

likely

mechanism

for

the

reassignments

in

platyhelminthes

and

echinoderms

The

process

would

have

begun

by

a

gain

in

function

of

the

tRNA-Asn

that

gave

it

some

ability

to

pair

with

AAA

and

hence

made

AAA

ambiguous

e.g.

the

Q

modification

ceased

to

occur

Then

further

changes

to

the

tRNA-Asn

would

have

increased

the

fraction

of

AAA

codons

translated

as

Asn

and

a

straightforward

mutation

of

the

tRNA-Lys

could

then

have

removed

this

ambiguity

Alternatively

it

is

possible

that

the

changes

occurred

via

the

UC

mechanism

If

the

tRNA-Lys

mutation

happened

first

this

would

leave

the

tRNA-Asn

able

to

pair

inefficiently

with

the

AAA

and

subsequent

changes

to

the

tRNA-Asn

would

allow

it

to

recognize

AAA

more

easily

This

argument

is

analogous

to

the

case

of

the

AUA

Ile

to

Met

change

or

the

AGR

Arg

to

Ser

However

in

those

two

cases

the

loss

of

function

is

the

deletion

of

the

original

tRNA

for

the

codon

in

question

which

is

irreversible

In

the

AAA

case

the

loss

of

function

is

just

the

mutation

of

U

to

C

in

the

anticodon

This

would

be

a

deleterious

mutation

that

could

easily

reverse

so

it

is

difficult

to

see

why

the

change

would

go

to

fixation

This

makes

the

UC

mechanism

seem

less

plausible

for

AAA

We

conclude

therefore

that

of

all

the

reassignments

considered

in

this

paper

these

two

examples

of

AAA

reassignment

are

the

best

candidates

for

the

AI

mechanism

In

the

case

of

Balanoglossus

the

AAA

codon

can

not

disappear

by

mutation

pressure

and

therefore

we

require

a

selective

reason

why

this

codon

should

be

absent

This

can

be

attributed

to

either

UC

or

AI

mechanisms

If

a

gain

of

function

of

the

tRNA-Asn

-LRB-

QUU

-RRB-

brought

about

by

the

loss

of

Q

modification

happened

first

the

resulting

tRNA-Asn

-LRB-

GUU

-RRB-

would

have

acquired

some

ability

to

decode

AAA

as

Asn

and

consequently

AAA

would

be

ambiguously

translated

The

negative

selection

against

such

ambiguous

translation

may

have

driven

the

AAA

codon

to

disappear

Subsequently

the

loss

of

function

of

tRNA-Lys

-LRB-

UUU

-RRB-

occurred

brought

about

by

a

mutation

from

U

to

C

at

the

first

anticodon

position

and

this

removed

the

ambiguity

in

AAA

decoding

Further

experimental

information

on

the

state

of

modification

of

the

tRNA-Asn

in

Balanoglossus

would

be

useful

to

confirm

this

Alternatively

the

loss

of

function

of

tRNA-Lys

that

prevents

it

from

translating

AAA

may

have

occurred

first

In

the

gain-loss

framework

this

would

be

a

loss

without

a

gain

AAA

would

be

unassigned

and

selection

against

this

codon

would

drive

its

usage

down

to

zero

This

is

analogous

to

the

situation

in

Schizosaccharomyces

octosporus

where

we

argued

that

the

AUA

codon

was

driven

to

zero

after

the

loss

of

the

tRNA-Ile

-LRB-

CAU

-RRB-

gene

However

this

case

is

again

made

less

plausible

by

the

fact

that

the

change

in

the

tRNA-Lys

would

be

reversible

whereas

the

loss

of

the

gene

from

S.

octosporus

is

irreversible

Indeed

the

current

situation

in

Balanoglossus

seems

unstable

A

mutation

of

the

tRNA-Lys

-LRB-

CUU

-RRB-

back

to

UUU

would

be

neutral

in

the

absence

of

AAA

codons

If

this

occurred

then

synonymous

mutations

of

AAG

codons

to

AAA

would

rapidly

occur

-LRB-

since

A

is

greater

than

G

-RRB-

This

would

drive

the

system

back

to

the

canonical

code

The

fact

that

AAA

remains

absent

in

Balanoglossus

suggests

that

there

is

some

reason

why

the

reversal

of

the

tRNA-Lys

back

to

UUU

would

not

be

neutral

One

possibility

may

be

that

CUU

pairs

more

efficiently

than

UUU

with

the

AAG

codons

so

translational

efficiency

would

favor

retention

of

the

CUU

anticodon

once

AAG

became

the

dominant

codon

for

Lys

Another

possibility

is

that

unmodified

wobble

position

U

bases

can

often

pair

with

all

four

bases

in

four

codon

families

For

this

reason

wobble

position

U

's

are

often

modified

when

they

occur

in

tRNAs

for

two-codon

families

only

-LRB-

Yokobori

et

al.

-RRB-

Thus

prior

to

the

codon

reassignment

the

tRNA-Lys

anticodon

would

be

U

UU

where

U

is

a

posttranscriptional

modification

of

U

that

permits

it

to

pair

with

A

and

G

only

If

the

ability

to

modify

the

U

were

lost

by

Balanoglossus

when

the

anticodon

mutated

to

CUU

then

a

reversal

of

this

mutation

back

to

UUU

would

leave

an

unmodified

U

in

the

wobble

position

that

could

pair

with

both

Asn

and

Lys

codons

This

would

be

selected

against

due

to

the

ambiguity

created

in

the

Asn

codons

Both

these

explanations

are

rather

speculative

Introduction

of

New

Stop

Codons

In

addition

to

the

AGR

block

which

becomes

Stop

in

vertebrates

there

are

two

further

cases

where

a

sense

codon

has

been

reassigned

to

Stop

According

to

the

GenBank

entry

-LRB-

G.

Burger

unpublished

-RRB-

UUA

has

been

reassigned

from

Leu

to

Stop

in

the

stramenopile

Thraustochytrium

aureum

-LRB-

Fig.

-RRB-

One

tRNA-Leu

gene

in

T.

aureum

has

a

CAA

anticodon

instead

of

the

usual

UAA

anticodon

hence

UUA

is

no

longer

translated

as

Leu

A

is

high

in

this

and

related

species

and

UUA

is

frequently

used

as

Leu

in

related

species

This

makes

it

unlikely

that

UUA

disappeared

due

to

mutation

pressure

The

mechanism

can

not

be

assigned

with

certainty

but

the

most

likely

explanation

is

that

UUA

was

driven

to

become

rare

after

the

mutation

in

the

tRNA-Leu

occurred

because

of

inefficient

pairing

with

the

CAA

anticodon

Gain

of

function

of

the

release

factor

could

then

have

occurred

This

scenario

corresponds

to

the

UC

mechanism

-LRB-

loss

before

gain

-RRB-

An

alternative

is

that

changes

in

the

release

factor

began

to

occur

in

such

a

way

that

UUA

became

ambiguously

translated

as

Leu

and

Stop

In

this

case

UUA

would

be

selected

against

as

a

Leu

codon

and

would

be

driven

to

low

frequency

and

selection

would

favor

the

mutation

to

C

in

the

anticodon

so

that

UUA

was

no

longer

translated

as

Leu

This

would

correspond

to

the

AI

mechanism

-LRB-

gain

before

loss

-RRB-

It

is

difficult

to

decide

between

these

scenarios

A

second

similar

case

is

the

reassignment

of

UCA

from

Ser

to

Stop

in

Scenedesmus

obliquus

-LRB-

Kück

et

al.

-RRB-

In

most

species

a

tRNA-Ser

-LRB-

UGA

-RRB-

would

decode

UCN

codons

but

in

S.

obliquus

the

tRNA-Ser

has

a

GGA

anticodon

that

can

only

pair

with

the

UCY

codons

UCA

has

become

the

standard

Stop

codon

used

in

the

standard

mitochondrial

protein

genes

Alongside

this

UCG

is

absent

presumably

because

it

no

longer

pairs

with

the

tRNA-Ser

The

Stop

codons

UAA

and

UGA

are

not

used

while

UAG

has

been

reassigned

to

Leu

-LRB-

as

discussed

above

under

reassignments

of

UAG

stop

codons

-RRB-

Table

shows

that

A

is

fairly

high

in

S.

obliquus

and

related

species

and

that

UCA

is

a

fairly

common

codon

in

both

P.

minor

and

C.

eugametos

where

it

retains

its

assignment

to

Ser

-LRB-

we

consider

C.

reinhardtii

separately

in

the

following

section

-RRB-

It

seems

unlikely

that

UCA

would

disappear

due

to

mutational

pressure

Here

again

it

is

possible

to

explain

the

result

in

terms

of

a

UC

or

an

AI

scenario

In

the

UC

scenario

the

tRNA-Ser

anticodon

would

mutate

to

GGA

which

would

drive

UCA

and

UCG

to

very

low

values

and

permit

the

release

factor

to

change

so

that

it

interacts

with

UCA

In

the

AI

scenario

the

release

factor

would

change

first

making

UCA

ambiguous

and

causing

it

to

be

selected

against

and

driven

to

low

frequency

and

allowing

the

mutation

in

the

tRNA-Ser

to

occur

Table

Codon

usage

in

some

green

algaeLeu

codonsIleSer

codonsThr

codonsGlu

codonsArg

codonsFFD

sitesUUAUUGAUAUCAUCGACAACGGAAGAGAGAAGG

A

GPedinomonas

Scenedesmus

Chlamydomonas

Chlamydomonas

Note

Intronic

and

other

nonstandard

ORFs

are

excluded

Unusual

selective

effects

influence

S.

obliquus

and

C.

reinhardtii

The

full

codon

usage

table

of

S.

obliquus

is

given

by

Kück

et

al.

-LRB-

-RRB-

and

Nedelcu

et

al.

-LRB-

-RRB-

In

Table

we

have

chosen

selected

codons

in

order

to

illustrate

that

S.

obliquus

is

unusual

in

other

ways

The

usual

Leu

UUA

codon

is

absent

and

the

tRNA-Leu

has

a

CAA

anticodon

which

translates

UUG

only

-LRB-

the

same

as

in

T.

aureum

except

that

UUA

is

not

used

as

a

Stop

codon

-RRB-

AGA

is

also

absent

but

there

is

a

tRNA-Arg

with

CCU

anticodon

that

translates

AGG

Nedelcu

et

al.

-LRB-

-RRB-

identified

a

tRNA-Arg

-LRB-

UCU

-RRB-

that

should

interact

with

AGA

and

a

tRNA-Ile

-LRB-

UAU

-RRB-

that

should

interact

with

AUA

However

AGA

and

AUA

are

both

absent

They

state

that

these

two

tRNAs

are

redundant

because

their

corresponding

codons

are

not

used

However

we

argue

that

these

two

tRNAs

must

be

nonfunctional

or

not

expressed

otherwise

the

corresponding

codons

would

be

used

Note

that

A

G

in

S.

obliquus

so

if

there

were

functional

tRNAs

with

U

at

the

wobble

position

the

A-ending

codons

would

be

frequent

As

an

illustration

the

Glu

codons

in

S.

obliquus

do

follow

the

expected

trend

from

the

mutational

frequencies

GAA

is

more

frequent

than

GAG

because

these

are

both

translated

by

a

tRNA-Glu

-LRB-

UUC

-RRB-

with

U

at

the

wobble

position

The

Thr

codons

shown

in

Table

also

have

ACA

more

frequent

than

ACG

but

one

final

peculiarity

of

the

S.

obliquus

genome

is

that

it

possesses

no

tRNA-Thr

It

is

presumed

that

a

tRNA-Thr

-LRB-

UGU

-RRB-

is

imported

from

the

cytoplasm

Importation

of

tRNAs

from

the

Cytoplasm

to

the

Mitochondria

Although

there

are

many

mitochondrial

genomes

that

possess

sufficient

tRNAs

to

translate

the

full

genetic

code

there

are

also

many

genomes

in

which

this

is

not

the

case

and

where

import

of

nuclear-encoded

tRNAs

from

the

cytoplasm

to

the

mitochondrion

must

be

occurring

Supplementary

Table

lists

the

amino

acids

for

which

tRNAs

are

present/absent

in

the

mitochondrial

genome

of

each

species

In

the

figures

we

have

used

a

white

arrow

symbol

to

indicate

positions

where

import

of

multiple

tRNA

genes

must

have

evolved

We

estimate

at

least

nine

independent

origins

of

tRNA

import

Acanthamoeba

Dictyostelium

Cnidaria

Chytridiomycota

land

plants

Pedinomonas

Chlamydomonas

Naegleria

Alveolata

and

kinetoplastids

-LRB-

the

latter

are

not

shown

but

are

known

to

use

tRNA

import

-RRB-

This

count

depends

on

the

details

of

the

phylogeny

it

is

possible

that

Acanthamoeba

and

Dictyostelium

have

a

common

ancestor

which

would

decrease

the

count

by

one

and

it

is

possible

that

Naegleria

is

not

a

sister

group

to

the

alveolates

which

would

increase

the

count

by

one

In

all

the

above

groups

at

least

four

tRNAs

are

absent

and

it

is

clear

that

the

remaining

tRNAs

are

insufficient

thus

there

is

no

doubt

that

import

must

occur

However

there

are

also

many

other

groups

where

only

a

small

number

of

tRNAs

are

missing

We

have

already

discussed

several

cases

above

where

the

loss

of

a

tRNA

leads

to

the

corresponding

codon

becoming

unassigned

or

reassigned

to

a

new

amino

acid

These

cases

leave

clear

signals

in

the

codon

usage

patterns

This

means

that

there

is

no

import

of

a

replacement

tRNA

from

the

cytoplasm

in

the

cases

discussed

in

previous

sections

In

contrast

we

now

discuss

several

cases

where

a

small

number

of

tRNAs

are

absent

from

the

mitochondrial

genomes

but

there

is

no

change

in

the

genetic

code

or

unusual

codon

usage

pattern

In

these

cases

it

appears

that

import

of

one

or

a

few

specific

tRNAs

is

occurring

There

is

no

tRNA

for

Thr

in

any

of

the

stramenopile

genomes

In

addition

tRNA-Thr

is

absent

from

Reclinomonas

the

Rhodophyta

Mesostigma

Scenedesmus

and

all

the

groups

where

multiple

tRNAs

are

missing

This

makes

tRNA-Thr

the

most

frequently

deleted

tRNA

We

consider

this

significant

but

we

have

no

plausible

explanation

why

The

tRNA-Thr

attracts

attention

because

there

are

many

genomes

in

which

this

is

the

only

tRNA

missing

Thr

codons

are

nevertheless

frequent

in

these

genomes

so

there

must

be

a

way

of

translating

them

Import

of

tRNA-Thr

from

the

cytoplasm

is

quite

possible

but

we

do

not

know

why

this

specific

tRNA

should

be

imported

Alternatively

in

the

genomes

where

tRNA-Thr

is

one

of

the

only

ones

missing

it

is

possible

that

another

tRNA

is

post-transcriptionally

edited

to

become

a

tRNA-Thr

Two

types

of

tRNA

would

thus

arise

from

the

same

gene

This

point

has

been

suggested

previously

-LRB-

Saks

et

al.

-RRB-

but

as

far

as

we

are

aware

there

is

no

direct

evidence

that

this

occurs

There

is

no

tRNA

for

the

Arg

CGN

block

in

several

of

the

stramenopiles

-LRB-

Cafeteria

Thraustochytrium

Chrysodidymus

Ochromonas

Laminaria

Pylaiella

-RRB-

but

it

is

present

in

Phytophthora

and

Saprolegnia

Complete

genomes

are

not

available

for

the

remainder

of

the

stramenopiles

shown

in

Fig.

hence

we

can

not

tell

if

the

gene

is

present

If

the

phylogeny

is

correct

this

implies

three

independent

losses

of

the

tRNA-Arg

Either

tRNA

import

or

some

other

tRNA

change

must

be

compensating

Recall

that

this

same

tRNA-Arg

is

also

absent

in

several

fungi

and

that

this

leads

to

the

CGN

block

becoming

unassigned

-LRB-

as

shown

in

Fig.

-RRB-

So

tRNA-Arg

is

not

imported

in

the

fungi

Supplementary

Table

lists

several

other

genomes

where

just

one

or

two

tRNAs

are

missing

It

is

possible

that

tRNA

import

occurs

or

that

there

are

unknown

tRNA

modifications

that

compensate

for

these

individual

losses

However

we

consider

this

as

somewhat

uncertain

in

cases

where

only

one

genome

is

known

with

the

missing

gene

This

could

simply

be

a

failure

to

locate

the

gene

on

the

genome

or

a

problem

of

misannotation

The

codon

usage

of

Chlamydomonas

reinhardtii

is

markedly

different

from

C.

eugametos

-LRB-

Table

and

Denovan-Wright

et

al.

-RRB-

Both

of

these

species

have

only

three

tRNAs

-LRB-

Supplementary

Table

-RRB-

hence

tRNA

import

is

required

C.

eugametos

uses

almost

all

codons

and

the

codon

usage

is

in

line

with

what

we

would

expect

from

the

FFD

site

frequencies

However

there

are

many

codons

that

are

absent

entirely

from

the

C.

reinhardtii

genome

This

suggests

that

a

restricted

set

of

tRNAs

is

being

imported

in

C.

reinhardtii

which

causes

selection

against

codons

that

can

not

be

translated

whereas

a

much

broader

set

is

being

imported

in

C.

eugametos

which

allows

a

more

standard

pattern

of

codon

usage

There

are

several

parallels

between

the

codon

usage

in

C.

reinhardtii

and

Scenedesmus

obliquus

-LRB-

a

sister

species

see

Fig.

-RRB-

UUA

-LRB-

Leu

-RRB-

AUA

-LRB-

Ile

-RRB-

UCG

-LRB-

Ser

-RRB-

and

AGA

-LRB-

Arg

-RRB-

are

all

absent

from

both

In

S.

obliquus

this

is

due

to

peculiarities

in

the

tRNAs

encoded

by

the

mitochondrial

genome

-LRB-

see

previous

section

-RRB-

The

correspondence

between

the

codon

usage

and

tRNA

set

in

S.

obliquus

shows

that

there

is

no

tRNA

import

in

this

species

It

therefore

appears

that

the

tRNAs

that

were

first

imported

in

C.

reinhardtii

correspond

to

those

that

were

needed

according

to

the

codon

usage

pattern

of

the

ancestor

of

C.

reinhardtii

and

S.

obliquus

In

C.

eugametos

the

evolution

of

tRNA

import

has

proceeded

further

A

more

complete

set

is

imported

and

the

codon

usage

has

relaxed

back

to

a

more

standard

pattern

due

to

mutation

Discussion

Distinguishing

the

Mechanisms

The

gain-loss

framework

that

we

use

to

classify

mechanisms

of

codon

reassignment

was

introduced

in

discussion

of

our

computer

simulations

-LRB-

Sengupta

and

Higgs

-RRB-

Here

we

wish

consider

to

what

extent

this

classification

is

applicable

to

the

real

events

The

CD

mechanism

is

defined

by

the

fact

that

the

codon

disappears

from

the

genome

and

is

replaced

by

synonymous

codons

prior

to

any

changes

in

the

tRNAs

and

release

factors

The

reasons

for

the

disappearance

are

random

drift

and

directional

mutation

pressure

In

simulations

it

is

obvious

whether

a

codon

has

disappeared

In

real

examples

there

is

clear

evidence

if

the

codon

usage

is

zero

or

close

to

zero

in

the

species

close

to

the

reassignment

point

and

there

is

also

clear

evidence

if

mutation

pressure

is

in

the

right

direction

to

cause

CD

It

is

of

course

never

possible

to

say

that

the

codon

usage

was

exactly

zero

in

some

genome

in

the

past

A

practical

definition

of

the

CD

mechanism

for

real

cases

is

that

absence

or

extreme

rarity

of

the

codon

is

the

major

factor

that

initiates

the

codon

reassignment

There

should

be

evidence

that

the

codon

was

rare

and

that

the

probability

of

CD

was

not

too

low

We

have

shown

that

this

is

true

in

the

cases

where

we

argue

that

the

CD

mechanism

occurred

Therefore

the

distinction

of

CD

from

the

alternatives

is

one

that

we

feel

can

be

reliably

made

in

real

examples

In

cases

where

we

have

determined

that

the

codon

did

not

disappear

the

central

question

is

to

distinguish

between

UC

and

AI

mechanisms

The

UC

mechanism

is

defined

by

the

fact

that

the

loss

occurs

before

the

gain

whereas

in

the

AI

mechanism

the

gain

occurs

before

the

loss

Although

this

distinction

is

obvious

in

simulations

it

can

be

more

difficult

in

real

cases

as

we

did

not

observe

the

order

of

events

Nevertheless

we

gave

several

examples

where

we

feel

a

reliable

classification

of

UC

can

be

made

In

particular

the

reassignments

of

AUA

from

Ile

to

Met

and

AGR

from

Arg

to

Ser

are

both

associated

with

the

deletion

of

a

tRNA

from

the

genome

The

loss

event

is

an

irreversible

gene

deletion

that

leaves

the

organism

in

a

deleterious

state

The

gain

event

is

then

positively

selected

in

response

to

this

loss

In

contrast

in

the

case

of

the

reassignment

of

AAA

from

Lys

to

Asn

the

loss

event

is

a

mutation

in

the

anticodon

of

the

tRNA-Lys

This

is

reversible

so

selection

would

favor

mutation

back

to

the

original

state

which

seems

more

likely

to

occur

than

making

a

codon

reassignment

The

gain

in

this

case

is

probably

due

to

the

cessation

of

the

Q

modification

process

which

could

occur

due

to

deleterious

mutations

in

the

modifying

enzyme

or

disruption

of

the

transport

of

the

enzyme

to

the

mitochondria

These

changes

seem

less

easily

reversible

than

a

single

mutation

in

the

anticodon

Therefore

we

argue

that

the

gain

occurred

first

and

that

the

loss

occurred

in

response

This

makes

the

AAA

reassignment

the

most

likely

example

of

the

AI

mechanism

The

cases

where

both

loss

and

gain

seem

easily

reversible

are

the

most

difficult

to

classify

and

both

AI

and

UC

scenarios

can

be

proposed

-LRB-

These

arguments

are

based

on

the

assumption

that

the

ambiguous

and

unassigned

states

are

deleterious

with

respect

to

the

original

code

We

deal

with

the

alternative

suggestion

that

ambiguous

translation

can

be

positively

selected

later

in

the

Discussion

-RRB-

We

just

argued

that

the

AUA

Ile-to-Met

case

is

initiated

by

deletion

of

the

tRNA-Ile

-LRB-

CAU

-RRB-

and

that

this

change

can

be

reliably

classed

as

UC

Nevertheless

it

is

possible

that

ambiguous

translation

plays

a

role

in

this

reassignment

in

the

following

sense

After

the

deletion

the

AUA

codon

would

be

translated

inefficiently

by

the

tRNA-Ile

-LRB-

GAU

-RRB-

but

it

is

possible

that

there

might

also

be

some

interaction

with

the

tRNA-Met

-LRB-

CAU

-RRB-

When

the

tRNA-Met

becomes

modified

it

definitely

gains

the

ability

to

translate

AUA

So

ambiguous

translation

of

AUA

as

both

Ile

and

Met

at

some

points

in

this

process

is

not

unlikely

Before

the

original

tRNA-Ile

-LRB-

CAU

-RRB-

is

deleted

any

slight

ability

of

the

other

two

tRNAs

to

translate

AUA

would

be

irrelevant

Also

after

the

modification

of

the

tRNA-Met

any

slight

ability

of

the

tRNA-Ile

-LRB-

GAU

-RRB-

to

translate

AUA

would

be

irrelevant

The

ambiguity

if

it

existed

would

only

be

relevant

when

there

is

a

competition

between

two

poorly

adapted

tRNAs

Despite

all

this

the

gain

in

function

of

the

tRNA-Met

only

occurs

after

the

loss

of

the

tRNA-Ile

-LRB-

CAU

-RRB-

so

this

reassignment

counts

as

UC

not

AI

We

recommend

the

use

of

the

term

AI

only

when

ambiguous

translation

occurs

as

a

result

of

a

gain

occurring

before

a

loss

and

where

competition

occurs

between

two

well-adapted

tRNAs

The

reassignment

of

AGR

from

Ser

to

Gly

in

urochordates

involves

a

gain

due

to

the

duplication

and

anticodon

mutation

of

the

tRNA-Gly

In

this

case

too

it

is

plausible

that

an

AI

stage

for

AGR

decoding

may

have

resulted

after

the

gain

of

the

new

tRNA-Gly

The

ambiguity

would

eventually

be

removed

due

to

the

selection

of

the

superior

recognition

of

AGR

by

the

new

tRNA-Gly

However

this

only

happens

after

the

deletion

of

the

original

tRNA-Arg

-LRB-

UCU

-RRB-

We

have

counted

the

changes

from

Arg

to

Ser

and

Ser

to

Gly

as

separate

events

because

the

amino

acid

changes

twice

However

we

may

think

of

the

duplication

of

the

tRNA-Gly

as

the

gain

that

finally

compensates

for

the

loss

of

the

tRNA-Arg

considerably

earlier

in

evolution

The

AGR

Ser

to

Gly

case

is

one

of

the

examples

where

the

number

of

tRNAs

increases

-LRB-

see

Table

and

discussion

below

-RRB-

but

this

only

returns

the

number

back

to

its

value

in

the

canonical

code

it

does

not

represent

an

increase

with

respect

to

the

canonical

code

Table

Summary

of

mechanisms

of

codon

reassignment

in

mitochondriaCodon

reassignmentNo

of

timesCan

this

be

explained

by

GC

AU

mutation

pressure?Change

in

no

of

tRNAsIs

mispairing

important?Reassignment

mechanismUAG

Stop

G

A

at

position

NoCDUAG

Stop

G

A

at

position

NoCDUGA

Stop

G

A

at

position

Possibly

CA

at

position.CDCGU

CGC/CGA/CGC

Arg

C

A

at

position

CUG

Leu

C

U

at

position

NoCDCUU/CUC/CUA

CUG

Thr

Ile

Met

or

GA

at

position.UCAAA

Lys

GA

at

position.AIAAA

Lys

GA

at

position.UC

or

AIAGA/AGG

Arg

GA

at

position.UCAGA

AGG

Ser

Ser

NoAIbUUA

Leu

or

AIUCA

Ser

or

AINote

CD

codon

disappearance

UC

unassigned

codon

AI

ambiguous

intermediate.aThe

loss

of

the

tRNA-Ile

has

occurred

three

times

-LRB-

once

in

metazoa

once

in

yeasts

and

once

in

S.

octosporus

-RRB-

but

the

reassignment

of

the

codon

to

Met

has

occurred

at

least

five

times

-LRB-

three

times

in

metazoa

and

twice

in

yeasts

-RRB-

bIf

AGR

was

fully

reassigned

to

Ser

prior

to

these

changes

then

they

are

probably

AI

However

they

may

also

be

considered

as

a

final

response

to

the

loss

of

the

original

tRNA-Arg

in

which

case

we

might

count

them

as

UC

A

slow

response

to

a

tRNA

deletion

occurs

for

both

the

tRNA-Ile

-LRB-

CAU

-RRB-

and

the

tRNA-Arg

-LRB-

UCU

-RRB-

which

are

deleted

at

roughly

the

same

time

in

the

ancestral

Bilateria

This

would

leave

AUA

inefficiently

translated

as

Ile

and

AGR

inefficiently

translated

as

Ser

Both

these

situations

remain

in

some

phyla

today

However

in

other

groups

AUA

has

been

captured

by

Met

and

AGR

either

has

become

a

useful

Ser

codon

-LRB-

due

to

mutation

of

the

tRNA-Ser

-RRB-

or

has

been

reassigned

to

Gly

or

Stop

These

secondary

changes

may

have

occurred

considerably

later

than

the

deletions

of

the

original

two

tRNAs

The

fourth

mechanism

occurring

in

the

gain-loss

framework

is

the

compensatory

change

mechanism

This

would

correspond

to

a

case

where

the

loss

and

gain

become

fixed

in

the

population

at

the

same

time

Organisms

with

either

the

gain

or

the

loss

but

not

both

would

only

occur

as

rare

variants

and

would

never

be

frequent

in

the

population

This

mechanism

is

expected

in

the

theory

due

to

the

analogy

of

the

problem

with

compensatory

mutation

theory

-LRB-

Kimura

Higgs

-RRB-

In

the

simulations

of

codon

reassignment

-LRB-

Sengupta

and

Higgs

-RRB-

it

was

possible

to

distinguish

a

few

cases

where

this

occurred

It

may

well

be

that

the

compensatory

change

mechanism

occurs

in

real

cases

and

it

could

be

an

explanation

in

cases

where

both

AI

and

UC

seem

equally

plausible

However

it

would

be

very

difficult

to

distinguish

this

from

either

the

UC

or

the

AI

mechanism

after

the

event

Comparison

with

Previous

Surveys

Recent

papers

by

Knight

et

al.

-LRB-

-RRB-

and

Swire

et

al.

-LRB-

-RRB-

have

also

carried

out

surveys

of

changes

in

the

mitochondrial

genetic

code

Knight

et

al.

-LRB-

-RRB-

highlighted

the

fact

that

variation

in

GC

content

plays

a

major

role

in

the

CD

mechanism

Most

mitochondrial

genomes

are

subject

to

mutation

pressure

from

GC

to

AU

Since

third

codon

positions

are

often

synonymous

they

argued

that

the

CD

mechanism

would

predict

that

the

codons

that

are

reassigned

should

have

C

or

G

at

the

third

position

As

this

is

not

always

the

case

they

argued

against

the

CD

mechanism

However

it

is

not

the

third

codon

position

per

se

that

matters

it

is

the

position

at

which

the

synonymous

mutation

occurs

Table

gives

examples

where

synonymous

mutations

driven

by

GC-to-AU

mutation

pressure

have

occurred

at

all

three

positions

Knight

et

al.

-LRB-

-RRB-

came

down

strongly

in

favor

of

the

AI

mechanism

We

wish

to

redress

the

balance

here

Our

data

show

the

CD

mechanism

is

the

best

explanation

in

over

half

the

cases

We

agree

with

Swire

et

al.

-LRB-

-RRB-

that

CD

applies

well

to

stop

codon

reassignments

but

does

not

usually

apply

to

sense

codon

reassignments

The

cases

of

CUN

and

CGN

reassignments

are

exceptions

to

this

where

we

argue

for

CD

with

sense

codons

In

their

proposal

of

the

AI

mechanism

Schultz

and

Yarus

-LRB-

-RRB-

identified

three

specific

types

of

codon-anticodon

mispairing

that

might

be

important

in

allowing

ambiguous

translation

of

a

codon

GA

at

the

third

codon

position

CA

at

the

third

codon

position

and

GU

at

the

first

codon

position

Knight

et

al.

-LRB-

-RRB-

considered

whether

these

mispairing

events

were

implicated

in

the

reassignments

in

mitochondrial

genomes

In

Table

we

also

do

this

-LRB-

cf.

Table

of

Knight

et

al.

-RRB-

We

agree

that

GA

mispairing

at

the

third

codon

position

is

important

in

the

reassignments

of

AAA

and

AGR

For

the

AUA

Ile-to-Met

transition

we

argue

that

GA

mispairing

between

tRNA-Ile

-LRB-

GAU

-RRB-

and

the

AUA

codon

is

important

otherwise

deletion

of

the

tRNA-Ile

-LRB-

CAU

-RRB-

would

not

be

possible

Mispairing

of

the

unmodified

tRNA-Met

-LRB-

CAU

-RRB-

with

AUA

-LRB-

as

in

the

Knight

et

al.

table

-RRB-

might

also

be

relevant

but

we

do

not

think

this

is

significant

prior

to

the

deletion

of

the

tRNA-Ile

-LRB-

CAU

-RRB-

The

reverse

transition

of

AUA

from

Met

back

to

Ile

listed

by

Knight

et

al.

does

not

occur

in

our

interpretation

-LRB-

Fig.

-RRB-

Also

the

reassignment

of

UAA

from

Stop

to

Tyr

listed

by

Knight

et

al.

is

no

longer

thought

to

occur

Knight

et

al.

argue

that

CA

mispairing

is

important

for

the

UGA

Stop

to

Trp

transition

There

is

evidence

for

this

in

Bacillus

subtilis

-LRB-

Lovett

et

al.

Matsugi

et

al.

-RRB-

where

UGA

is

ambiguous

It

is

not

clear

whether

this

is

a

key

feature

in

the

UGA

reassignments

in

mitochondria

There

are

many

species

where

the

canonical

system

works

fine

-LRB-

UGA

is

Stop

UGG

is

Trp

and

the

tRNA-Trp

has

CCA

anticodon

-RRB-

It

is

possible

that

if

CA

mispairing

started

to

occur

due

to

some

change

in

the

tRNA-Trp

outside

the

anticodon

UGA

would

become

ambiguous

and

this

would

provide

a

selective

pressure

against

UGA

codons

that

would

help

to

drive

them

to

disappear

However

we

are

not

aware

of

any

evidence

for

this

in

mitochondria

The

disappearance

of

UGA

is

likely

to

occur

in

any

case

due

to

mutation

pressure

and

drift

and

this

seems

to

be

a

more

important

factor

The

other

cases

listed

by

Knight

et

al.

where

mispairing

is

implicated

do

not

occur

in

mitochondria

and

we

did

not

consider

them

From

Table

we

would

argue

that

mispairing

is

important

in

some

but

not

all

reassignments

However

the

existence

of

mispairing

does

not

necessarily

imply

the

AI

mechanism

occurred

and

mispairing

is

not

a

diagnostic

feature

of

AI

as

previously

envisioned

If

translation

of

a

codon

becomes

inefficient

due

to

tRNA

loss

then

selection

will

act

against

it

and

it

will

tend

to

be

replaced

by

synonymous

codons

If

this

situation

remains

for

some

time

without

any

compensating

gain

of

function

in

another

tRNA

then

the

codon

may

in

fact

disappear

We

have

argued

that

this

is

the

case

in

S.

octosporus

where

the

tRNA-Ile

-LRB-

CAU

-RRB-

has

been

deleted

and

AUA

has

disappeared

and

also

in

the

two

Kluyveromyces

species

in

which

AUA

is

very

rare

-LRB-

Table

-RRB-

A

similar

thing

happens

with

the

AGR

reassignment

there

are

several

invertebrate

groups

in

which

AGG

becomes

rare

after

the

deletion

of

the

tRNA-Arg

because

of

inefficient

pairing

with

the

tRNA-Ser

when

it

retains

its

original

GCU

anticodon

-LRB-

Table

-RRB-

In

cases

where

there

is

a

full

gain

in

function

of

the

new

tRNA

the

codon

in

no

longer

selected

against

This

is

seen

when

modifications

occur

in

both

the

tRNA-Met

and

the

tRNA-Ser

Given

that

the

loss

of

the

tRNA

in

the

first

step

of

the

UC

mechanism

is

likely

to

be

deleterious

anything

that

offsets

the

deleterious

effect

to

some

extent

will

increase

the

likelihood

of

the

UC

mechanism

occurring

It

has

been

suggested

that

deletion

of

a

tRNA

gene

might

be

favored

since

it

reduces

the

total

length

of

the

genome

and

hence

allows

more

rapid

replication

-LRB-

Andersson

and

Kurland

-RRB-

We

considered

this

effect

in

our

simulations

-LRB-

Sengupta

and

Higgs

-RRB-

and

showed

that

selection

for

reduction

in

genome

length

does

indeed

increase

the

likelihood

of

the

UC

mechanism

This

factor

will

be

more

important

in

smaller

genomes

because

the

relative

change

in

length

for

deleting

a

tRNA

will

be

larger

For

the

cases

of

deletion

of

the

tRNA-Ile

and

tRNA-Arg

that

we

are

considering

we

do

not

have

an

experimental

measurement

of

the

selective

disadvantage

to

the

translation

system

of

loss

of

the

gene

so

it

is

not

possible

to

say

whether

the

potential

advantage

of

shortening

the

genome

is

large

or

small

compared

to

this

We

do

not

wish

to

argue

that

these

reassignments

are

caused

by

selection

to

reduce

the

genome

size

but

we

do

wish

to

say

that

it

is

the

chance

deletion

of

these

tRNAs

that

initiated

the

reassignment

in

these

cases

and

that

the

fixation

in

the

population

of

the

genome

with

the

deletion

could

well

be

aided

by

selection

for

more

rapid

genome

replication

However

there

is

no

general

trend

for

tRNA

deletion

at

codon

reassignment

We

have

given

the

changes

in

tRNA

number

associated

with

our

interpretation

of

the

reassignment

events

in

Table

There

are

three

cases

of

tRNA

gain

each

of

which

occurs

once

These

are

due

to

a

tRNA

duplication

followed

by

an

anticodon

mutation

There

are

four

cases

of

tRNA

deletion

which

together

occur

times

Changes

in

release

factors

are

not

included

in

this

table

because

these

genes

are

not

coded

by

the

mitochondrial

genome

In

Table

in

the

supplementary

information

of

Swire

et

al.

-LRB-

-RRB-

a

distinction

is

made

between

the

theoretical

and

the

observed

changes

in

tRNA

number

Given

the

genome

information

now

available

this

distinction

is

no

longer

necessary

The

AGR

Arg

to

Ser

reassignment

in

Bilateria

is

classed

as

an

observed

gain

by

Swire

et

al.

-LRB-

their

case

-RRB-

because

they

are

comparing

with

Cnidaria

However

it

is

clear

that

tRNA

import

has

evolved

separately

in

Cnidaria

and

that

it

does

not

occur

in

either

Porifera

or

the

ancestral

Bilaterian

The

proper

comparison

for

Bilateria

is

with

Porifera

where

the

tRNA-Arg

is

present

therefore

it

is

evident

that

the

gene

has

been

deleted

in

Bilateria

at

the

point

of

the

codon

reassignment

Case

of

Swire

et

al.

is

the

AUA

Ile

to

Met

that

occurs

in

yeasts

We

have

shown

in

this

paper

that

the

tRNA-Ile

-LRB-

CAU

-RRB-

gene

is

present

in

the

fungi

prior

to

this

reassignment

and

is

deleted

at

the

point

of

the

reassignment

Once

again

this

is

a

clear

deletion

and

there

is

no

difference

between

the

observed

and

the

expected

change

An

important

issue

in

the

AI

mechanism

is

the

nature

of

the

selective

effect

on

an

ambiguous

codon

In

our

model

-LRB-

Sengupta

and

Higgs

-RRB-

we

assumed

that

ambiguous

translation

would

be

unfavorable

because

the

wrong

amino

acid

would

be

inserted

part

of

the

time

However

it

has

been

suggested

that

ambiguous

translation

may

be

favorable

-LRB-

Santos

et

al.

-RRB-

Swire

et

al.

-LRB-

-RRB-

also

gave

an

argument

related

to

the

cost

of

amino

acid

biosynthesis

whereby

ambiguous

translation

might

be

favorable

because

the

savings

in

the

cost

of

using

an

energetically

cheaper

amino

acid

might

outweigh

the

penalty

of

the

ambiguity

The

ambiguity

penalty

may

be

very

small

at

particular

sites

where

the

amino

acid

substitution

is

neutral

or

almost

neutral

hence

the

cost

savings

would

make

this

change

advantageous

This

mechanism

is

possible

and

we

are

currently

in

the

process

of

studying

it

using

simulations

of

the

same

type

as

those

used

by

Sengupta

and

Higgs

-LRB-

-RRB-

A

problem

we

see

with

this

argument

is

that

if

an

amino

acid

substitution

were

favored

by

cost

saving

it

would

be

easier

to

achieve

this

by

a

straightforward

mutation

in

the

gene

without

changing

the

genetic

code

Such

mutations

could

occur

in

places

where

it

was

favorable

and

not

in

places

where

it

was

not

There

must

be

some

sites

in

the

protein

where

the

substitution

is

deleterious

even

if

there

are

also

some

neutral

sites

On

the

other

hand

if

the

codon

were

reassigned

this

would

force

changes

in

all

sites

in

the

protein

whether

favorable

or

not

which

would

act

against

the

postulated

benefit

of

the

change

We

do

not

think

that

positive

selection

for

ambiguous

translation

can

be

a

general

explanation

of

sense-to-sense

reassignments

We

have

already

given

one

case

where

a

sense-to-sense

reassignment

is

better

explained

by

the

CD

mechanism

and

several

where

it

is

better

explained

by

UC

Swire

et

al.

-LRB-

-RRB-

did

not

distinguish

between

AI

and

UC

However

the

cost-savings

argument

is

less

relevant

for

UC

than

AI

In

AI

if

the

cost-savings

scenario

occurred

it

would

be

central

to

the

argument

because

it

would

provide

a

reason

why

the

gain

occurred

first

i.e.

it

would

be

the

driving

force

for

the

reassignment

In

the

UC

mechanism

the

cause

of

the

transition

is

the

chance

loss

event

which

is

presumed

to

be

deleterious

and

is

not

driven

by

selection

The

gain

event

would

then

be

positively

selected

because

it

would

get

rid

of

the

penalty

from

the

slow

translation

that

occurs

when

there

is

no

well-adapted

tRNA

The

cost-savings

argument

might

also

favor

the

gain

event

in

this

case

This

would

change

the

details

of

the

selective

forces

favoring

the

gain

but

this

would

only

be

relevant

after

the

initial

deletion

However

there

is

no

need

to

postulate

the

existence

of

the

neutral

sites

and

cost

savings

in

the

UC

case

because

the

gain

would

be

driven

by

selection

to

improve

translational

efficiency

anyway

Thus

if

the

cost-savings

scenario

occurred

in

a

UC

reassignment

it

would

be

a

minor

detail

not

a

driving

force

In

any

case

the

best-documented

example

of

ambiguous

translation

of

a

codon

is

the

CUG

codon

in

the

Candida

nuclear

genome

-LRB-

Santos

et

al.

-RRB-

and

this

example

now

provides

evidence

that

ambiguous

translation

is

deleterious

It

has

been

shown

that

the

reassignment

is

driven

by

the

gain

of

a

new

tRNA-Ser

with

anticodon

CAG

that

translates

the

CUG

codon

which

is

normally

Leu

This

case

was

formerly

cited

as

an

example

of

positive

selection

for

ambiguous

translation

However

Massey

et

al.

-LRB-

-RRB-

have

now

shown

that

there

is

selection

against

the

ambiguous

CUG

codons

that

causes

them

to

become

very

rare

in

their

original

Leu

positions

Most

occurrences

of

CUG

in

Candida

are

newly

evolved

in

Ser

positions

This

shows

that

there

is

negative

selection

on

the

ambiguous

codon

-LRB-

Note

that

this

is

the

third

reason

for

a

codon

to

become

rare

in

the

CD

mechanism

the

codon

disappears

due

to

mutation

pressure

in

the

UC

mechanism

the

codon

is

inefficiently

translated

and

selected

against

and

therefore

might

become

rare

in

the

AI

mechanism

the

codon

might

be

selected

against

because

it

is

ambiguous

and

therefore

might

become

rare

-RRB-

Another

study

suggesting

that

ambiguous

translation

is

deleterious

is

that

by

Kim

et

al.

-LRB-

-RRB-

who

studied

many

examples

of

anticodon

mutations

in

tRNA-Phe

from

E.

coli

In

cases

where

the

cells

expressing

the

mutant

tRNA

were

viable

it

was

found

that

the

mutant

tRNA

was

charged

with

the

amino

acid

corresponding

to

the

new

anticodon

i.e.

there

was

no

ambiguous

translation

However

in

other

cases

expression

of

the

mutant

tRNA

was

lethal

It

was

presumed

that

the

mutant

tRNA

was

still

charged

with

Phe

although

its

anticodon

corresponded

to

another

amino

acid

This

would

cause

ambiguous

translation

which

was

apparently

deleterious

in

the

experiment

The

arguments

in

this

paper

apply

only

to

codon

reassignments

that

have

occurred

since

the

establishment

of

the

canonical

code

They

do

not

apply

to

the

origin

and

buildup

of

the

canonical

code

We

believe

that

there

is

a

good

case

that

positive

selection

was

important

in

the

establishment

of

the

canonical

code

-LRB-

Freeland

et

al.

Ardell

and

Sella

-RRB-

and

that

this

accounts

for

the

nonrandomness

and

apparent

optimization

of

the

code

Adding

a

new

amino

acid

to

the

code

opens

up

a

whole

new

realm

of

protein

sequences

that

can

be

formed

using

the

new

amino

acid

Therefore

there

is

ample

scope

for

positive

selection

to

act

in

determining

the

codons

to

which

each

amino

acid

is

initially

assigned

Translational

error

rates

and

mutation

rates

may

have

been

large

in

early

organisms

so

minimizing

the

effects

of

these

errors

may

have

been

important

However

we

do

not

think

that

positive

selection

guides

the

codon

reassignments

that

have

occurred

after

the

establishment

of

the

canonical

code

There

is

little

to

be

gained

from

a

codon

reassignment

in

modern

organisms

because

no

new

amino

acid

is

being

added

and

the

effects

of

minimizing

error

will

be

very

small

when

both

translational

error

and

mutation

rates

are

as

small

as

they

are

today

tRNA

Evolution

Although

Table

shows

no

strong

trend

to

reduce

the

number

of

tRNAs

at

codon

reassignment

events

we

feel

that

this

obscures

the

very

large

role

that

tRNA

deletion

has

had

in

the

evolution

of

the

mitochondrial

translation

system

A

major

difference

between

bacterial

and

mitochondrial

systems

is

that

four-codon

families

in

bacteria

require

at

least

two

tRNAs

one

with

wobble

position

G

to

translate

U

and

C-ending

codons

and

one

with

wobble

position

U

to

translate

A

and

G-ending

codons

Most

mitochondria

require

only

one

tRNA

with

wobble

position

U

for

all

four

codons

In

metazoan

mitochondria

the

second

tRNA

has

been

deleted

in

all

eight

four-codon

families

The

same

genes

have

been

deleted

in

other

groups

too

and

we

suspect

that

there

have

been

independent

deletions

of

the

same

gene

although

demonstrating

this

would

be

difficult

because

we

do

not

have

a

fully

resolved

tree

for

the

earliest

branches

of

the

eukaryotes

Nevertheless

the

same

process

has

occurred

at

least

once

in

all

eight

four-codon

families

which

is

relevant

to

the

discussion

on

genome

streamlining

even

though

there

is

no

change

in

the

code

Loss

of

a

tRNA

with

wobble

position

G

may

have

been

slightly

deleterious

originally

when

it

occurred

in

the

early

stages

of

mitochondrial

evolution

but

it

is

possible

that

the

ribosome

has

become

sufficiently

flexible

to

tolerate

this

It

could

also

be

that

the

loss

still

comes

with

a

price

of

decreased

accuracy

and/or

speed

of

translation

Selection

for

reduction

in

genome

length

might

well

play

a

role

in

these

deletions

also

The

tRNAs

in

animal

mitochondria

represent

the

minimal

possible

set

It

is

only

possible

to

reduce

the

number

further

by

making

codons

unassigned

or

by

evolving

a

mechanism

of

tRNA

import

from

the

cytoplasm

We

have

seen

that

tRNA

import

has

evolved

multiple

times

If

import

is

possible

then

there

will

be

no

deleterious

effect

of

further

tRNA

deletions

from

the

mitochondrial

genome

and

selection

for

shorter

genome

length

may

favor

the

fixation

of

variants

in

which

tRNAs

have

been

deleted

Thus

rapid

tRNA

deletion

might

be

expected

in

lineages

in

which

a

general

import

process

is

operating

It

is

known

that

many

protein

genes

formerly

present

on

the

mitochondrial

genome

have

been

transferred

to

the

nuclear

genome

-LRB-

Blanchard

and

Lynch

-RRB-

Many

of

these

code

for

proteins

that

are

required

in

the

mitochondrion

and

are

targeted

back

to

the

organelle

after

synthesis

In

cases

where

tRNAs

are

imported

it

is

interesting

to

ask

whether

these

tRNAs

are

in

fact

formerly

mitochondrial

tRNA

genes

that

have

been

transferred

to

the

nucleus

or

whether

the

existing

nuclear

tRNAs

have

become

functional

in

the

mitochondria

and

the

mitochondrial

genes

have

simply

been

deleted

We

attempted

to

answer

this

question

with

the

kinetoplastids

Trypanosoma

brucei

and

Leishmania

tarentolae

which

possess

no

mitochondrial

tRNAs

and

where

import

is

known

to

occur

-LRB-

Simpson

et

al.

Hancock

and

Hajduk

Schneider

and

Marechal-Drouard

-RRB-

The

full

set

of

tRNAs

in

the

nuclear

genome

of

these

species

is

known

We

carried

out

phylogenetic

analysis

comparing

these

genes

with

tRNAs

from

other

eukaryotic

nuclei

bacteria

and

mitochondria

For

each

separate

tRNA

the

kinetoplastid

genes

clustered

with

eukaryotic

nuclear

genes

and

not

with

mitochondria

We

do

not

show

these

results

because

the

trees

have

very

low

statistical

support

due

to

the

short

sequence

length

of

the

tRNAs

Nevertheless

we

found

no

evidence

that

any

of

the

kinetoplastid

tRNAs

had

been

transferred

from

the

mitochondrial

genome

The

identity

elements

and

antideterminants

of

tRNAs

are

key

aspects

of

the

molecules

that

play

a

crucial

role

in

the

proper

aminoacylation

and

prevention

of

misacylation

of

a

tRNA

-LRB-

Giegé

et

al.

-RRB-

These

are

important

factors

in

understanding

changes

in

the

genetic

code

Most

aminoacyl

tRNA-synthetases

use

the

anticodon

as

a

key

identity

element

for

proper

recognition

of

the

tRNA

Two

exceptions

are

Leu

and

Ala

where

the

anticodon

has

no

role

to

play

in

the

proper

charging

of

the

cognate

tRNA

with

the

amino

acid

In

such

cases

mutations

in

the

anticodon

do

not

affect

the

aminoacylation

of

the

tRNA

Such

mutations

can

result

in

codon

reassignment

if

the

resulting

anticodon

acquires

the

ability

to

pair

with

a

codon

originally

associated

with

a

different

amino

acid

The

reassignment

of

UAG

from

Stop

to

Leu

in

the

mitochondrial

genome

of

some

species

of

green

plants

provides

such

an

example

-LRB-

Laforest

et

al.

-RRB-

Another

possibility

is

that

misacylation

of

a

tRNA

due

to

a

change

in

an

aminoacyl

tRNA-synthetase

could

be

a

cause

of

a

codon

becoming

ambiguous

The

tRNA

would

be

ambiguously

charged

but

there

would

only

be

one

type

of

tRNA

for

the

codon

However

this

does

not

seem

to

occur

in

the

mitochondrial

reassignments

we

considered

here

Conclusions

We

have

given

arguments

above

as

to

which

mechanisms

seem

most

likely

in

each

of

the

codon

reassignments

in

mitochondrial

genomes

We

have

shown

that

the

many

reassignments

of

stop

codons

to

sense

codons

are

readily

explained

by

CD

given

the

biased

base

composition

of

most

mitochondrial

genomes

and

the

small

total

number

of

occurrences

of

stop

codons

in

these

genomes

Disappearance

of

sense

codons

is

more

difficult

because

the

total

number

of

codons

for

the

corresponding

amino

acid

is

large

A

very

strong

mutational

bias

is

required

for

sense

codons

to

disappear

However

in

one

group

of

yeast

species

an

extreme

bias

against

C

does

exist

and

we

argue

that

the

reassignment

of

CUN

and

CGN

codons

in

these

species

is

attributable

to

CD

In

the

other

examples

of

sense

codon

reassignments

the

mutational

bias

is

in

the

wrong

direction

for

causing

CD

and

the

probability

of

disappearance

is

negligible

Where

the

codon

does

not

disappear

we

have

emphasized

the

important

distinction

between

the

UC

and

AI

mechanisms

The

case

for

the

UC

mechanism

is

most

clear

when

the

reassignment

is

associated

with

a

tRNA

deletion

We

then

argue

that

the

deletion

initiated

the

process

and

the

codon

reassignment

occurred

as

a

response

to

this

The

UC

mechanism

does

not

rely

on

selection

for

reducing

genome

length

but

if

such

selection

were

significant

this

would

increase

the

likelihood

of

this

mechanism

Many

nonessential

tRNAs

have

been

deleted

during

mitochondrial

genome

evolution

and

these

did

not

initiate

codon

reassignments

because

the

original

code

was

still

functional

after

the

deletion

However

this

makes

it

clear

that

chance

tRNA

deletion

is

a

relatively

common

event

We

also

observed

several

cases

where

a

good

argument

can

be

made

for

the

AI

mechanism

i.e.

where

the

reassignment

arose

because

the

codon

first

became

ambiguous

In

other

cases

scenarios

for

both

AI

and

UC

seemed

equally

plausible

and

it

is

difficult

to

distinguish

them

after

the

event

We

see

these

genetic

code

changes

as

chance

events

rather

than

as

changes

governed

by

positive

selection

Disappearance

is

a

chance

event

that

occurs

under

drift

when

there

is

strong

mutational

bias

in

base

frequencies

If

a

change

in

tRNAs

or

release

factors

happens

to

occur

while

a

codon

is

absent

then

a

codon

reassignment

can

occur

However

this

is

a

chance

event

the

codon

frequency

could

drift

back

to

a

higher

level

without

any

reassignment

occurring

Our

interpretation

of

reassignments

via

UC

and

AI

mechanisms

is

that

they

too

are

initiated

by

chance

events

such

as

the

deletion

of

a

tRNA

gene

or

a

change

in

the

process

of

base

modification

in

an

anticodon

These

changes

are

probably

slightly

deleterious

but

efficient

functioning

of

the

translation

system

can

be

restored

by

making

the

codon

reassignment

The

view

that

AI

states

are

driven

by

positive

selection

seems

unlikely

to

us

at

present

The

origin

of

the

canonical

code

is

outside

the

scope

of

the

present

paper

However

we

emphasize

that

the

situation

in

codon

reassignments

in

modern

organisms

is

different

from

that

during

the

early

evolution

of

the

canonical

code

where

positive

selection

probably

had

an

important

role

We

conclude

that

our

gain-loss

framework

is

suitable

as

a

description

of

the

real

codon

reassignment

events

It

emphasizes

that

there

are

several

mechanisms

that

are

alternatives

within

a

larger

picture

and

that

it

is

not

always

profitable

to

discuss

these

mechanisms

as

though

they

were

mutually

exclusive

These

mechanisms

can

and

do

occur

in

nature

and

one

mechanism

is

not

sufficient

to

explain

all

cases

Electronic

Supplementary

Material

Supplementary

material

codon reassignment
mitochondrial genetic codes
unassigned codon mechanism
ambiguous intermediate mechanism