第24回 蛋白質科学会年会
WS19 モ ル生物大腸菌における翻訳制御研究の最前線

大腸菌由来の再構成型無細胞系内の翻訳反応は大腸菌細胞と同一か?
金森 崇、布施(村上) 朋重、松本 令奈（ジーンフロンティア(株)）2024. 6. 13

で

デ

1

PURE system
PUREfrex is based on the PURE system technology.
The PURE system is a reconstituted cell-free protein synthesis system, which consists only of puri ed factors
necessary for transcription, translation and energy regeneration in Escherichia coli.

Shimizu Y. et al. (2001) Nat. Biotechnol., vol. 19, p. 751-755.
Shimizu Y. et al. (2005) Methods, vol. 36, p. 299-304.
fi

2Topics★

PURE system (Translation in E. coli)★

Initiation in the PURE system★

Optimum sequence for the PURE system‣ 5’-UTR‣ N-terminal region in the ORF

3

Topics★

PURE system (Translation in E. coli)★

Initiation in the PURE system★

Optimum sequence for the PURE system‣ 5’-UTR‣ N-terminal region in the ORF

4

PURE system
PUREfrex is based on the PURE system technology.
The PURE system is a reconstituted cell-free protein synthesis system, which consists only of puri ed factors
necessary for transcription, translation and energy regeneration in Escherichia coli.

Shimizu Y. et al. (2001) Nat. Biotechnol., vol. 19, p. 751-755.
Shimizu Y. et al. (2005) Methods, vol. 36, p. 299-304.
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5

Initiation
MTF (Methionyl-tRNA transformylase)ATP• transfers formyl group from 10-formyl-tetrahydrofolic acid (10-CHO-THF) to the free

MetRS

Met

Met

OHC MetMTF

amino group in methionine bound to initiator tRNA

IF1 (Initiation Factor 1)

10-CHO-THF• binds the A-site in the 30S subunit and prevents binding of aminoacyl-tRNA.

Initiator tRNA• modulates and stabillizes binding of IF2 and IF3 to the 30S subunit.

IF2
GTP

IF2 (Initiation Factor 2)• is a GTPase and hydrolyzes GTP to GDP during formation of the 70S initiation complex.

IF1• binds formylmethionyl-initiator tRNA and promotes it the entry to the P-site.

IF3• is expressed as isoforms (α, β, β’) from in-frame di erent AUG codons of infB gene.

OHC MetIF3 (Initiation Factor 3)
5’

SD

3’

AUG

mRNA

• binds to the 30S subunit and prevents association of the premature 50S subunit.
• enhances the accomodation of formylmethionyl-initiator tRNA to the P-site.

70S initiation complex• Its gene contains the non-canonical initation codon AUU.

All of four proteins are essential for cell growth.
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6Elongation
ATP
AA

EF-Tu (Elongation Factor Tu)

ARS• delivers aminoacyl-tRNA to the A-site of ribosome in a GTP-boud form.
AA• is encoded by two genes, tufA and tufB.

tRNA
EF-Tu

EF-Ts (Elongation Factor Ts)

GTP• associates GDP-bound EF-Tu and stimulate the exchange of GDP and GTP.

EF-Ts

EF-G (Elongation Factor G)AA5’• facislitates the translocation of the ribosome in a GTP hydrolysis-dependent manner.
3’

NNN NNN NNNmRNA

All of three EFs are essential for cell growth.

EF-G
GTP

AA

5’

3’

NNN NNN NNN

mRNA
7

Elongation
ATP
AA

EF-Tu (Elongation Factor Tu)

ARS• delivers aminoacyl-tRNA to the A-site of ribosome in a GTP-boud form.
AA• is encoded by two genes, tufA and tufB.

tRNA
EF-Tu
EF-P

EF-Ts (Elongation Factor Ts)

GTP• associates GDP-bound EF-Tu and stimulate the exchange of GDP and GTP.

EF-Ts

EF-G (Elongation Factor G)
ABC-F
ATP
5’

AA

• facislitates the translocation of the ribosome in a GTP hydrolysis-dependent manner.
3’

NNN NNN NNN

mRNA

EF-P and ABC-F proteinsEF-G• promotes the formation of the peptide bond for the speci c sequences.

GTP

EF-P: poly-Pro
YheS: SecM, Uup: poly-Pro, YbiT: K/E repeat and D/E repeat, EttA: D/E repeat

AA5’

Ude et al. (2013) Science, 339, 82-85.
Doerfel et al. (2013) Science, 339, 85-88.

3’

NNN NNN NNN

Murina et al. (2019) J. Mol. Biol., 431, 3568-3590.

mRNA

Chadani et al. (2024) Nucleic Acids Res., gkae309.

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8Elongation (E ect of EF-P)
AmiB (E. coli)

IL6 (mature) (H. sapiens)

129

1

Lys34

MVPPGEV P P P P P P P PV

kDa

kDa
100

Oriole staining

100
75

75
50

50

AmiB
(45.6 kDa)

37

PDB: 3A5Z

37

EF-P
IL6 (23.1 kDa)
IL6_short

25

25

EF-P

20
15

20

15

Western blotting with anti-FLAG

10

EF-P (µM)

00.03 0.3
0.1
1
0.01

EF-P modification

3

0.03
0.3
0.01 0.1
1

3
EF-P (µM)

0

0.1

0.3

1

3

0.1

0.3

1

3

IL6
IL6_short

EF-P modification

ff

EF-P enhanced the synthesis of proteins with Pro-Pro sequence in the PURE system.

Termination
RF1 (Release Factor 1)
RF1• recognizes the stop codon UAG and UAA, and releases the nascent polypeptide chain.

RF2• is post-translationally methylated at the position Gln 235 in the conserved GGQ motif.

RF2 (Release Factor 2)

RF3• recognizes the stop codon UGA and UAA, and releases the nascent polypeptide chain.

GTP
5’

NNN NNN

3’

UAA

mRNA

• is post-translationally methylated at the position Gln 252 in the conserved GGQ motif.
• Its gene contains UGA codon at the position 26 and the full-length protein issynthesized by +1 frameshft.
• Substitution of Thr at the position 246 to Ala increases the termination e ciency.

RF3 (Release Factor 3)• is a ribosome-dependent GTPase.
• promotes the release of RF1 and RF2 from the ribosome after the release of thepeptide chain.
5’

NNN NNN

3’

UAA

mRNA

RF1 and RF2 are essential for cell growth.
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10Termination
RF1 (Release Factor 1)
RF1• recognizes the stop codon UAG and UAA, and releases the nascent polypeptide chain.

RF2• is post-translationally methylated at the position Gln 235 in the conserved GGQ motif.

RF2 (Release Factor 2)

RF3• recognizes the stop codon UGA and UAA, and releases the nascent polypeptide chain.

GTP
5’

NNN NNN

3’

UAA

mRNA

• is post-translationally methylated at the position Gln 252 in the conserved GGQ motif.
• Its gene contains UGA codon at the position 26 and the full-length protein issynthesized by +1 frameshft.
• Substitution of Thr at the position 246 to Ala increases the termination e ciency.

RF3 (Release Factor 3)• is a ribosome-dependent GTPase.
• promotes the release of RF1 and RF2 from the ribosome after the release of thepeptide chain.
5’

NNN NNN

3’

UAA

mRNA

RF1 and RF2 are essential for cell growth.
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Recycling
RRF (Ribosome Recycling Factor)• Is involved in ribosome recycling with EF-G after the termination process.

RRF is essential for cell growth.

5’

NNN NNN

3’

UAA

mRNA
RRF
EF-G
GTP

5’

NNN NNN

3’

UAA

mRNA
12

PUREfrex

Shimizu Y. et al. (2001) Nat. Biotechnol., vol. 19, p. 751-755.
Shimizu Y. et al. (2005) Methods, vol. 36, p. 299-304.
13

PUREfrex
Example of protein synthesis

14Topics★

PURE system (Translation in E. coli)★

Initiation in the PURE system★

Optimum sequence for the PURE system‣ 5’-UTR‣ N-terminal region in the ORF

15

Initiation in the PURE system (MTF and 10 -CHO-THF)
MetRS

Met

OHC MetMTF

10-CHO-THF
Met

MTF

10-CHO-THF

Initiator tRNA
IF2

IF1
IF3
OHC Met

5’

SD

3’

AUG

mRNA
70S initiation complex

Formylation of initiator methionine was not essential
for the translation in the PURE system.

16

Initiation in the PURE system (IFs)
MetRS

Met

OHC MetMTF

IF1, IF2, IF3
Met

10-CHO-THF

Initiator tRNA
IF2

IF1
IF3
OHC Met

5’

SD

3’

AUG

mRNA
70S initiation complex

Initiation factors were not always required
for the translation reaction.

17

Initiation in the PURE system (initiation codon)
MetRS

Met

Met

sfGFP

OHC MetMTF

M S K G E E ..
atgtctaaaggtgaagaa..

10-CHO-THF

Initiator tRNA
IF2

IF1

mCherryIF3

M A I I K E ..
atggctattattaaagaa..

OHC Met

5’

SD

3’

AUG

mRNA
70S initiation complex
Initiation codons in E. coli

(Ref; Blattner et al. (1997) Science, 277, p. 1453)

AUG; 83%
GUG; 14% (e.g. EF-Tu, ArgRS, HisRS, RRF)
UUG; 3%ffi

18

AUU; 2 genes (infC (IF3), pcnB)

GTG, TTG, and ATT functioned as the initiation codon, but the synthesis
e ciency was dependent on the target proteins.

Summary
Initiation in the PURE system
All
IFs
and
formylationof
initiator
methioninearenotessentialforthe‣translation in the PURE system, but IF2 and IF3 enhance the translatione ciency.
Codonsusedasinitiation
codon
in
E.
coli
are
availableforthe‣translation in the PURE system, albeit with low e ciency.

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19

Topics★

PURE system (Translation in E. coli)★

Initiation in the PURE system★

Optimum sequence for the PURE system‣ 5’-UTR‣ N-terminal region in the ORF

20

5’ UTR of the template DNA for PUREfrex
Currently used sequence (derived from T7 gene 10 UTR)

Stemloop region is necessary fore cient transcription reaction andstabilization of the transcript.
AT-rich region binds S1 protein of
30S ribosome subunit and increases
translation e ciency.
SD (Shine-Dalgarno) sequence binds 3’terminus of 16S rRNA and localizes
mRNA to the start position of translation.

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SD and Spacer

Long SD sequence< 10 nt spacer
Currently used SD sequence

5-12 nt spacer
Short SD sequence

8-10 nt spacer

22

currently usedsequence (control)Stemloop

currently usedsequence (control)

Stemloop region

23

3 nt

6 nt

Stemloop and AT-richcurrently usedsequence (control)

6nt-SL
AT-rich region24

No< 12 nt> 15 nt

E ect of deletion of 5’ UTR of the downstream gene(+) AT-rich/SD(-) AT-rich(-) SD(-) AT-rich/SD(-) linker

SD sequence was required, but AT-rich region was not, for translation of the downstream gene.

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25

E ect of deletion of 5’ UTR of the upstream gene(+) AT-rich/SD(+) AT-rich(-) AT-rich(+) AT-rich

Short SD(+) AT-rich(-) SD(+) AT-rich(+) AT-rich/SD(-) AT-rich(-) AT-rich(-) AT-rich

Short SD(-) AT-rich(-) SD(-) AT-rich

Deletion of AT-rich region or SD sequence in the upstream gene increased translationof the downstream gene only with AT-rich region in the 5’ UTR.
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26

Summary
5' UTR for the translation in the PURE system

6 nt for e cient transcription

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27

essential for translation

essential for translation> 12 nt for e cient translation> 3 nt for e cient translation

Summary

control
SL-AT10ta
6nt-AT21
6nt-AT15at

28

Topics★

PURE system (Translation in E. coli)★

Initiation in the PURE system★

Optimum sequence for the PURE system‣ 5’-UTR‣ N-terminal region in the ORF

29

E ect of the sequence on the protein synthesis
Example of protein synthesis

Synthesis e ciency is highly dependent on the target proteins.
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30

E ARTICLE
ect of N-terminal sequence on the protein synthesis
OPEN

https://doi.org/10.1038/s41467-019-13810-1

A short translational ramp determines the
ARTICLEOPEN Peptidyl transferase center
efﬁciency of protein synthesis
AManasvi
shorttranslational
determines
the Lavagnino , Erica N. Thomas ,Verma, Junhong Choi ramp, Kyle
A. Cottrell , Zeno
decompaction
andstructural
efﬁciencyof protein
synthesis, PawelSzczesny, David W. Piston , Hani S. Zaher , Joseph D. Puglisi&
Slavica Pavlovic-Djuranovic
Sergej Djuranovic *constraints during early protein
elongation on the ribosome
OPEN

https://doi.org/10.1038/s41467-019-13810-1

1,8

2,3,6,8

1

1

Manasvi Verma1,8, Junhong Choi

1234567890():,;

1,7

5

1

4

4

2

2,3,6,8, Kyle A. Cottrell1,8, Zeno Lavagnino1,7, Erica N. Thomas4,

Slavica Pavlovic-Djuranovic1, Pawel Szczesny
Sergej Djuranovic 1*1234567890():,;

1,8

5, David W. Piston

1, Hani S. Zaher4, Joseph D. Puglisi

2 &Translation initiation is a major rate-limiting step for protein synthesis. However, recent
Translation
initiation is suggest
a major rate-limitingfor protein
recentstudies strongly
that the step
efﬁciency
ofsynthesis.
protein However,synthesis
is additionally regulated bystudies strongly suggest that the efﬁciency of protein synthesis is additionally regulated bymultiple factors that impact the elongation phase. To assess the inﬂuence of early elongation
multiple factors that impact the elongation phase. To assess the inﬂuence of early elongation
proteinsynthesis,
we aemployeda libraryof morethan
250,000
onon
proteinsynthesis,
we employedlibrary of morethan 250,000
reporters
combinedwith reporters combined with
inin
vitro
and andin vivoinprotein
assays. Here weassays.
report thatthe identityof the amino
vitro
vivo expression
protein expression
Here
we reportthat the identity of the aminoacids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNA
acids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNAabundance, translation initiation efﬁciency, or overall mRNA structure. Single-molecule
abundance,of translationtranslation
initiation
efﬁciency,
or overallmRNAstructure. Single-molecule
measurements
kinetics
revealed pausingof the ribosome
and aborted
proteinsynthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions. Finally,measurements of translation kinetics revealed pausing of the ribosome and aborted protein
introduction of preferred sequence motifs only at speciﬁc codon positions improves protein
synthesison codons
4 and
5 of Collectively,
distinct amino
and thenucleotide
compositions. Finally,
synthesis
efﬁciency
for recombinant
proteins.
our data acidunderscore
criticalroleof early elongation
events in translational
controlof geneexpression.
introductionof preferred
sequence
motifsonly
at speciﬁc codon positions improves protein
synthesis efﬁciency for recombinant proteins. Collectively, our data underscore the criticalrole of early elongation events in translational control of gene expression.

Verma et al. (2019) Nat. Commun.

Bin Jia1, Tianlong Wang1* & Jean Lehmann2*
Peptide bond formation on the ribosome requires that aminoacyl-tRNAs and peptidyl-tRNAs areproperly positioned on the A site and the P site of the peptidyl transferase center (PTC) so that
nucleophilic attack can occur. Here we analyse some constraints associated with the induced-fit
mechanism of the PTC, that promotes this positioning through a compaction around the aminoacyl
ester orchestrated by U2506. The physical basis of PTC decompaction, that allows the elongatedpeptidyl-tRNA to free itself from that state and move to the P site of the PTC, is still unclear. Fromthermodynamics considerations and an analysis of published ribosome structures, the present work
highlights the rational of this mechanism, in which the free-energy released by the new peptide
bond is used to kick U2506 away from the reaction center. Furthermore, we show the evidence that
decompaction is impaired when the nascent peptide is not yet anchored inside the exit tunnel, which
may contribute to explain why the first rounds of elongation are inefficient, an issue that has attracted
much interest for about two decades. Results in this field are examined in the light of the present
analysis and a physico-chemical correlation in the genetic code, which suggest that elementary
constraints associated with the size of the side-chain of the amino acids penalize early elongation
events.

Jia et al. (2021) Sci. Rep.

During protein synthesis on the ribosome, the peptidyl-transferase center (PTC) sequentially catalyzes theformation of new peptide bonds between incoming aminoacyl tRNAs and a growing peptide passed on fromtRNA to tRNA. Unlike most enzymes, the ribosome has to deal with a large variety of substrates in the formof aminoacyl-tRNA, where the L side-chain of the aminoacyls can be absent (glycine) or very bulky (e.g. tryptophane), and exhibit extreme properties in terms of charge distribution and hydrophobicity. It implies thatno direct interaction between the PTC and the side chain could occur to orient an aminoacyl for nucleophilic
attack. Instead, the PTC achieves this positioning through an induced-fit mechanism1 that creates a compaction
around the Cα of the aminoacyl ester, freezing it in an appropriate orientation almost without interfering withthe side chain2. As the compact state has only recently been recognized as such, the issue of PTC decompaction
after peptide bond formation, that allows the A-site peptidyl tRNA product to get free and move to the P site,
has not been investigated so far.
Based on a survey and examination of ribosome structures complexed with various arrest peptidyl-tRNAand peptidyl-tRNA analogs retrieved from the pdb database, and from free-energy estimates, the present workshows that some of the free-energy released by the reaction of peptide bond formation is used to decompact the

N-terminal 3rd to 5th amino acid residues are important for e cient translation.
1 Department of Cell Biology and Physiology, Washington University School of Medicine, 600 South Euclid Avenue, Campus Box 8228, St. Louis, MO 63110,

31USA. 2 Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126, USA. 3 Department of Applied Physics, Stanford
University, Stanford, CA 94305-5126, USA. 4 Department of Biology, Washington University, St Louis, MO 63105, USA. 5 Department of Bioinformatics,
Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland. 6Present address: Department of Genome Sciences, University of

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8

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Larger amino acids, which are encoded by AT-rich codon, facilitated early elongation.

N-terminal codon suitable for PUREfrex
N-terminal codon of Trastuzumab HC

AT-rich codons > Major codons
32

Optimum sequence of the template DNA for PUREfrex
5’UTR (from T7 phage gene10)
T7 promoter

StemloopAT-richSD

GGGAGA(for transcription)≧ 15 bases(essential)≧ 3 bases(essential)

ORF

AT-rich codons, not major codons
Multiple codons, not just major codons
No slippery sequence33

E ect of temperature on the protein synthesis
Fuse-Murakami et al. (2024) Int. J. Mol. Sci., 25, 5264.

PUREfrex 2.1 (4 mM GSH)
+ template DNA
incubationfor 24 hours
at 23, 30 or 37ºC
SDS-PAGE

Synthesis e ciency at lower temperature depended on the target proteins.

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E ect of 4th amino acid on sfGFP synthesis
sfGFP

M
S
K
G
E
E
L
F
T
G
ATGTCTAAAGGTGAAGAATTATTTACTGGT..
T
TTT
T

C

A

TTC

ff

TAT

TCC

TAC

TCA

TTG
CTT
CTC

Leu

Ser

Stop

TCG

TAG

Stop

TGG

Trp

CCT

CAT

CCC

CAC

Pro

CCG

CAG

ATT

ACT

AAT

ACC

AAC

ATA

ACA

Thr

CAA

AAA

ACG

AAG

GTT

GCT

GAT

GTC

GCC

GAC

GTA

Met

Val

TGC

Cys

TGA

CTG
Ile

Tyr

TGT

Stop

CCA

ATC

G

TAA

CTA

GTG
35

Phe

A

TCT

TTA

ATG

G

C

GCA
GCG

Ala

GAA
GAG

His
Gln
Asn
Lys
Asp
Glu

CGT
CGC
CGA

Arg

CGG
AGT
AGC
AGA
AGG

Ser
Arg

GGT
GGC
GGA
GGG

Gly

E ect of 4th amino acid on sfGFP synthesis
Fuse-Murakami et al. (2024) Int. J. Mol. Sci., 25, 5264.

sfGFP

PUREfrex 2.1 (4 mM GSH)
+ template DNA
incubationfor 24 hours
at 23, 30 or 37ºC

M
S
K
G
E
E
L
F
T
G
ATGTCTAAAGGTGAAGAATTATTTACTGGT..

23ºC

30ºC

37ºC

Fluorescence of sfGFP at 24 h(% of control)

200

100

0

Y
tat

H
cat

N
aat

P
cct

S
tct

I
att

T
act

L
ctt

D
gat

F
ttt

R
cgt

C
tgt

A
gct

S
agt

G
ggt

V
gtt

The fourth amino acid was very important for the e cient translation at lower temperature.
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36

E ect of 4th amino acid on sfGFP synthesis
Fuse-Murakami et al. (2024) Int. J. Mol. Sci., 25, 5264.

sfGFP

PUREfrex 2.1 (4 mM GSH)
+ template DNA
incubationfor 24 hours
at 23, 30 or 37ºC

M
S
K
G
E
E
L
F
T
G
ATGTCTAAAGGTGAAGAATTATTTACTGGT..

Fluorescence of sfGFP at 24 h(% of control)

23ºC

30ºC

37ºC
control

100

0

tat

tac
Y

aat

aac
N

tct

tcc

tca

tcg
S

agt

agc

tta

ttg

ctt

ctc
L

cta

ctg

gct

gca

gcc
A

gcg

ggt

gga

ggc
G

At the fourth position, the in uence of amino acids was greater than that of synonymous codons.

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37

ggg

E ect of N-terminal tag on the protein synthesis
PUREfrex 2.1 (4 mM GSH)
+ template DNA
incubationfor 24 hours
at 23, 30 or 37ºC
SDS-PAGE

Luc : MEDAKNIKKG..
ALP: MRTPEMPVLE..
DHFR: MISLIAALAV..

Fuse-Murakami et al. (2024) Int. J. Mol. Sci., 25, 5264.

ALP

SKG/SKY

Amount of synthesized ALP(% of EF-Tu)

100

23 ºC

30 ºC

37 ºC

50

0

WT

(+) SKG(+) SKY

N-terminal additional sequence increased the amount of synthesized ALP at all temperature.

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38

Expression of sfGFP variants in E. coli
BL21(DE3) + pET-sfGFP DNA(Tyr/Asn/Ala/Gly@4th)
Incubation at 23, 30 or 37°C
+ IPTG (at OD=1)
Incubation at 23, 30 or 37°C
Measure OD and uorescence

Fuse-Murakami et al. (2024) Int. J. Mol. Sci., 25, 5264.

Fluorescence

Synthesis E ciency
in the PURE system

High

∆OD

Low

High

Low

Variants that were highly synthesized in the PURE system were also highly expressed in E. coli cells.
The di erence in the expression level between variants was less than that in the PURE system.
Expression of Ala and Gly variants suppressed the growth of E. coli cells.

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Summary
Optimum sequence for the translation in the PURE system
AT-richcodonsarepreferable
inthe
N-terminal
region.
‣Multiplecodonsshould
be
used
for
each
amino
acid
in
the
entire
ORF.
‣
Amino
acids
at
the
N-terminal
regionarevery
important
for
e
cient
‣
protein synthesis especially at lower temperature.

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Summary
Translation in the PURE system = Translation in E. coli cells ?
Initiation
factorsarenotessential.
‣
AT-richregion
in
the
5'UTR
is
important
as
well
as
SD
sequence.
‣
Translationeciencyatlower
temperatureisdependentonthesequence‣of the target proteins.

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Summary
Translation in the PURE system ≈ Translation in E. coli cells ?
Initiation
factorsarenotessential.
‣
AT-richregion
in
the
5'UTR
is
important
as
well
as
SD
sequence.
‣
Translationeciencyatlower
temperatureisdependentonthesequence‣of the target proteins.

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