MOLECULAR RECOGNITION AND EVOLUTION OF Escherichia coli TYROSINEtRNA BY TYROSYL-tRNA SYNTHETASE
1

2

2

1

1

1, 2

Haruichi Asahara , Jun Iwaki , Junji Yokozawa , Koji Tamura , Nobukazu Nameki and Tsunemi Hasegawa
12Institute of Space and Astronautical Science, Sagamihara, Kanagawa 229-8510, Japan

Department of Material and Biological Chemistry, Faculty of Science, Yamagata University,
Yamagata 990-8560, Japan
Phone/Fax: +81-23-628-4604
E-mail: hasegawa@sci.kj.yamagata-u.ac.jp(Received 28 July 2005

Accepted 29 August 2005)

Present address:
Haruichi Asahara: Center for Molecular Biology of RNA, University of California,
Santa Cruz, CA 95064, USA
Koji Tamura: The Scripps Research Institute, La Jolla, CA 92037, USA
Nobukazu Nameki: Faculty of Engineering, Gunma University, Kiryuu 376-8515, Japan(Abstract)
Identity determinants of Escherichia coli tRNA

Tyr

were examined using in vitro transcripts. The first and second

anticodon nucleotides were base-specifically involved in tRNA recognition of tyrosyl-tRNA synthetase, whereas
the third anticodon nucleotide was not. None of the identity determinants were found in the acceptor stem,
except the discriminator base A73. With respect to the long variable arm, a stem of three base pairs in length
was required for tyrosylation, while the sequence of the arm was not essential. From the results of identity
conversion experiments, the discrimination modes of tRNAsome of the identity determinants of tRNA

Tyr

Tyr

from other amino acid specific tRNAs sharing

were discussed.

(Keywords)
TyrtRNA ; tRNA identity; tyrosyl-tRNA synthetase; Escherichia coli

Viva Origino 33 (2005) 194 - 207© 2005 by SSOEL Japan- 194 -

Viva Origino 33 (2005) 194 - 207

Tyr

1. IntroductiontRNA

The correct attachment of amino acid to tRNAs by its

five nucleotides in the variable region [12]. Thiscognatestructural difference seems to be responsible for theaminoacyl-tRNA

synthetase(aaRS)

isbelongs to class I tRNAs, which have four or

Tyrimportant for the precise translation of genetic

difference in the way that tRNA

information in protein synthesis. An aaRS musttyrosyl-tRNA synthetase (TyrRS) between organisms.

discriminate its cognate tRNAs from the pool of

Saccharomyces

tRNAs having a similar overall tertiary structure. The

aminoacylate Escherichia coli tRNAsequence and structural features of a tRNA that

In contrast, E. coli TyrRS does not aminoacylate S.

define this discrimination are named "identity

cerevisiae amber suppressor tRNAdeterminants". A number of studies using several

Although tRNA identity in the tyrosine system isgenetic, biochemical and biophysical approaches

interesting from an evolutionary point of view, data

have shown that many aaRSs recognize a small set ofof the identity determinants is still incomplete. In E.

nucleotides in the corresponding tRNA, which arecoli, in vivo and in vitro mutational studies

often located in the anticodon, the fourth base fromdemonstrated that the first and second anticodon

the 3' terminus subsequent to the universal CCA3'

nucleotides and the discriminator base A73 aresequence (the discriminator base) and the acceptor

involved in aminoacylation with tyrosine [14-16].

stem [1-6]. In addition to these positive identity

However, other nucleotides, including the third

determinants important for recognition by cognate

anticodon nucleotide A36, have not yet been closely

aaRS, negative identity determinants were found to

examined. As for the long variable arm, thefunction in the prevention of misacylation by non-mutational study has suggested the importance of its

cognate aaRSs [6-11].

direction for tyrosylation [16]. However, the role of

tRNAs

are

dividedintotwogroupsaccording to the length of the variable region. This

Tyr

Tyr

along with tRNA

Ser

Leu

and tRNA , possesses a long

TyrRS
Tyr

Tyr

does

not

in vitro [13].

in vivo [13].

the length and the sequence of the long variable armin tyrosylation remain unclear.
In this study, we constructed various tRNA

classification is conserved throughout kingdoms in
all tRNAs except tRNA . In eubacteria, tRNA ,cerevisiae

is recognized bymutanttranscripts,

andmeasuredtheiraminoacylation kinetics with E. coli TyrRS in vitro invariable arm composed of more than ten nucleotides,

order

and these three tRNAs are classified as class II

determinants

tRNAs. In eukaryotes and archaebacteria, however,conversion experiments from both class I (Lys and- 195 -

tosystematicallyofexamineTyrtRNA .

theidentity

Moreover,identityViva Origino 33 (2005) 194 - 207

Asp) and class II (Ser and Leu) tRNAs were

Toyopearl 650, Tosoh, Tokyo). The TyrRS fraction

performed

TyrRS

had a specific activity of 56 U/mg (One unit offrom the other tRNAs. On theaminoacyl-tRNA synthetase activity was defined as

in

order

discriminates tRNA

Tyr

to

clarifyhowdeterminants that enables tRNA

Tyr

Tyr

identity

the amount of the enzyme that catalyzes thediscrimination

incorporation of 1 nmol of amino acid into tRNA inbasis of the results obtained, a set of tRNA

10 min under the reaction conditions describedfrom other tRNAs was determined.

below.). The aminoacylation reaction proceeded at
2. Materials and Methods

37 oC in 30 µl of reaction mixture containing 60 mM

2.1. Preparation of template DNAs and in vitro

Tris-HCl (pH 7.5), 10 mM magnesium chloride, 2transcripts

mM dithiothreitol, 0.1 mg/ml bovine serum albumin,

Synthetic DNA oligomers carrying the T7 promoter

2.5 mM ATP, 11 µM L-[U- C] tyrosine (17.6

and tRNA genes were ligated into pUC19 andGBq/mmol), and various concentrations of tRNAtransformed into E. coli strain JM109. The templatetranscripts and E. coli TyrRS. The initial rates ofDNAaminoacylation

sequenceswereconfirmedbydideoxy

14

were determined by using

six

sequencing. Transcripts of the tRNA genes wereconcentrations of tRNA transcripts ranging from 0.1

prepared in a reaction mixture containing 40 mM

to 10 µM at a fixed concentration of the synthetase,

Tris-HCl (pH 8.1), 5 mM dithiothreitol, 2 mM

depending on the mutant tRNA transcripts. The Km

spermidine, 10 mM magnesium chloride, 50 µg/ml

and Vmax values were determined from Lineweaver-bovine serum albumin, 2 mM each NTP, 20 mM

Burk plots with the use of linear least-squares

5'GMP, BstNI-digested template DNA (0.2 mg/ml), 2

analysis. The Vmax/Km values for two or three

U of inorganic pyrophosphatese (Sigma), and T7

independent determinations were within ±15 %.

RNA polymerase (50 µg/ml) that had been purified
from the overproducer E. coli BL21/pAR1219 [17].

3. Results

The

To investigate the effects of the base modification ontranscripts

werepurified

by

15

%

polyacrylamide gel electrophoresis.

tyrosylation, the kinetic parameter of the native
tRNA

Tyr

was compared with that of the wild-type
Tyr

Tyr

2.2. Aminoacylation assay

sequence of the tRNATyrRS was partially purified from E. coli strain Q13

has

by anion exchange column chromatography (DEAE-

hypermodified G, queuosine (Q), at the position of- 196 -

seven

modifiedtranscript. E. coli tRNA
nucleotides

includingaViva Origino 33 (2005) 194 - 207

the first anticodon nucleotide. As shown in Table 1,
the native tRNA

Tyr

Tyr

tRNA s. Since the nucleotides that are not preserved
Tyrpossessed a Vmax/Km value only

among these five tRNA s do not base-specifically

1.5-fold larger than that of the transcript, indicating

contribute to aminoacylation by E. coli TyrRS, a basethat this bulky modification, together with other baseor a base-pair substitution was introduced into themodifications, is not essential for recognition by E.

positions of the conserved nucleotides among thesecoli TyrRS.

tRNAs.

According to cross-species aminoacylation
experiments,

E. coli

TyrRS canaminoacylate

Tyr3.1. Acceptor stemtRNA s from Bacillus stearothermophilus, Bacillus

In the acceptor stem, G2, G5-C68 and G6 are

subtilis, Neurospora crassa mitochondria, and yeast

conserved

mitochondria

aminoacylatedashomogeneous tRNAefficiently
Tyr

as

those

from

among
by

E.

Tyr

heterogeneoustRNA s

coli

(Fig.

TyrRS

1).

[18]. As shown in Figure 1,

Substitutions of G2-C71 by C2-G71, G5-C68 by C5-

except for invariant nucleotides only limited numbers

G68 and G6-C67 by C6-G67 resulted in decreases inof nucleotides were conserved among these five

the Vmax/Km value by 1.7-, 3.1-, and 4-fold,

Tyr

Fig. 1. Consensus sequences of tRNA

from E. coli, Bacillus stearothermophilus, Bacillus subtilis, Neurospora
Tyrcrassa mitochondria and yeast mitochondria. These tRNA s can be aminoacylated by E. coli TyrRS. Additional
Tyrnucleotides in mitochondrial tRNA are indicated by closed circles. Invariant or semi-invariant bases are designatedby outlined letters. This figure is cited from [18].

- 197 -

Viva Origino 33 (2005) 194 - 207

TyrFig. 2. The cloverleaf structure of E. coli tRNA with the base modifications omitted. Numbering is according to[12]. Arrows indicate the substitutions made in this study.

respectively (Fig. 2, Table 1). These findings indicate

nucleotides in tyrosylation in detail, each nucleotidethat these three base pairs in the acceptor stem are

was substituted by the other three nucleotides (Fig. 2).

not involved in tyrosylation. U9, located between the

The substitution of the first anticodon nucleotide G34

acceptor stem and the D arm, is not only conservedby the other three nucleotides decreased Vmax/KmTyramong heterogeneous tRNA s aminoacylated by E.
coli TyrRS, but is also characteristic of tRNA

Tyrvalues by 25- to 56-fold (Table 1). The kinetic dataof the C34 mutant were approximately in agreement

because the other class II tRNAs possess G9.

with those reported by Hou & Schimmel [15]. The

However, the substitution of G9 for U9 had only aeffects of mutation on tyrosylation appeared mostslight effect on tyrosylation activity (Fig. 2, Table 1).

drastically when the mutation occurred at the second

Thus, we concluded that the identity determinants ofanticodon nucleotide. The substitution of U35 by

TyrtRNA , except the discriminator base A73, do not

A35 reduced the Vmax/Km value 3100-fold (Table

reside in the acceptor stem.

1). The other substitutions, U35 by G35 and U35 by
C35, decreased the Vmax/Km value 240- and 670fold, respectively

3.2. Anticodon
To

study

the

involvement

of

the

anticodon(Table 1).

In contrast,

thesubstitution of the third anticodon nucleotide A36 by- 19 -

Viva Origino 33 (2005) 194 - 207

Table 1
Tyr

Kinetic parameters with E. coli tyrosyl-tRNA synthetase for tRNA transcripts______________________________________________________________________
Km

Vmax

Vmax/Km

loss of(µM)(nmol/min(relative)

specificity

per mg)(relative)______________________________________________________________________Tyr

Native tRNA

0.34

5.6

1.5

0.53

5.9

1.0

transcript
tRNA

Tyr

Tyr

tRNA (C2-G71)
TyrtRNA (C5-G68)

1

1.4

8.9

0.58

1.7

1.0

3.7

0.32

3.1

Tyr

3.3

9.2

0.25

4

Tyr

0.58

4.4

0.67

1.5

tRNA (C6-G67)
tRNA (G9)TyrtRNA (G47-C47d)

1.1

9.5

0.81

1.2

1.7

0.91

0.048

21

-

-

<0.0001>10000tRNA (A34)

2.3

1.0

0.040

25

Tyr

tRNA (C34)

3.6

0.79

0.020

50

Tyr

1.9

0.38

0.018

56

67

0.24

0.00032

3100

TyrtRNA ∆(2)TyrtRNA ∆(3)
TyrtRNA (U34)
TyrtRNA (A35)
TyrtRNA (G35)
TyrtRNA (C35)
Tyr

5.9

0.28

0.0042

240

22

0.38

0.0015670

tRNA (G36)

0.52

2.4

0.42

2.4

tRNA (C36)

Tyr

0.91

5.3

0.52

1.9

Tyr

2.8

7.8

0.25

4

tRNA (U36)______________________________________________________________________

Numbered nucleotides and base pairs in parentheses refer to the substitutions oftRNAs (Fig. 2).
∆(2) means the deletion of two base pairs; 46-47e, 47-47d, and ∆ (3) means
the deletion of three base pairs; 45-47f, 46-47e, 47-47d, in variable arm
Tyrof tRNA transcripts.

any other nucleotide decreased the Vmax/Km values

3.3. Variable arm

only a few-fold (Table 1). These results indicate thatIn the variable arm, only C47-G47d is conserved

the first and second anticodon nucleotides are base-among heterogeneous tRNA s aminoacylated by E.

specifically required for aminoacylation by TyrRS,

coli TyrRS (Fig. 1). Substitution by G47-C47d did

whereas the third anticodon nucleotide is not.

not affect the tyrosylation activity (Fig. 2, Table 1).

Tyr- 199 -

Viva Origino 33 (2005) 194 - 207

Tyrcontains three Watson-

increased the tyrosylation activity up to 1/13 (Table

Crick base pairs, which form the variable stem. The

2). These results indicate that the tRNA -type

deletion of one base pair reduced the Vmax/Km

anticodon nucleotides and discriminator base arevalue 21-fold (Fig. 2, Table 1). Additional one-base-sufficient to allow the discrimination of tRNApair deletion more severely decreased tyrosylationfrom the other class II tRNAs.

The variable arm of tRNA

Tyr

activity (Fig. 2, Table 1). These results indicate that a

Tyr

Identity conversion experiments were also
Lys

Asp

variable arm with a stem of three base pairs in length

applied to class I tRNAs, tRNA

is required for tyrosylation, but that a specific

3(c), 3(d)). Wild-type tRNAsequence is not.

U34U35U36 and A73, and thus among the major

Lysidentity determinants of tRNATyr

and tRNA(Fig.

transcript contains(excepting the long

3.4. Identity conversion

variable arm) only the first anticodon nucleotide is

The above and previous results indicated that thedifferent from that of tRNA . Although the wild-

anticodon nucleotides and the discriminator base aretype tRNATyrcrucial identity determinants of tRNA . Here, theTyrtRNA -type

anticodon

nucleotides

anddiscriminator base were introduced into tRNA
tRNA

Leu

Ser

Tyr

Lys

transcript had no tyrosylation activity,

the substitution of U34 by G34 increased thetheVmax/Km value up to a detectable level, 1/2100

andcompared with that of tRNAto clarify to what extent these elements are

involved in tRNATyr

discrimination from the otherfor tRNA

Asp

Tyr(Table 2). Likewise,

containing G34U35C36 and G73, only

the discriminator base is different from that of
Tyr

Asp

class II tRNAs (Fig. 3(a), 3(b)). These transcriptstRNA . The wild-type tRNAwith a wild-type sequence were not charged withcharged with tyrosine (Table 2). The substitution ofSertranscript was nottyrosine (Table 2). For tRNA , the change of both

G73 by A73 slightly decreased the Vmax/Km value

the anticodon nucleotides from U34G35A36 to

up to 1/3600 (Table 2). To investigate whether the

G34U35A36, and the discriminator base from G73 tothird anticodon nucleotide is important in thisA73,

discrimination mode, C36 of this tRNA

increased

theVmax/Km

value

of

the

tyrosylation activity up to 1/45 compared with that ofwild-type tRNA

Tyr

Leu

Asp

mutantwas changed to A36. This additional mutation(Table 2). In case of tRNA ,improved the Vmax/Km value by only about 5-foldwhich possesses the same discriminator base as(Table 2). These results show that the substitution of

TyrtRNA , the conversion of the anticodon nucleotides

the anticodon nucleotides and the discriminator basefrom C34A35G36 to G34U35A36 alone dramaticallyalone does not confer efficient tyrosylation activity- 200-

Viva Origino 33 (2005) 194 - 207

on class I tRNAs. It seems most likely that the longsecond anticodon nucleotides and the discriminatorvariable arm plays an important role in thebase.

discrimination by TyrRS in addition to the first andSer

Fig. 3. The transcripts for the identity conversion from tRNA
indicate the substitutions made in this study.

Leu(a), tRNA- 201-

Lys(b), tRNA

Asp(c), and tRNA(d). Arrows

Viva Origino 33 (2005) 194 - 207

Table 2Kinetic parameters with E. coli tyrosyl-tRNA synthetase for tRNA transcripts______________________________________________________________________
Km

Vmax

Vmax/Km

loss of(µM)(nmol/min(relative)

specificity

per mg)(relative)______________________________________________________________________
tRNA

Tyr

0.53

5.9

1.0

tRNA

Ser

-

-

<0.0001>10000

Ser

1.3

0.31

0.022

45

-

-

<0.0001>10000

2.6

0.22

0.079

13

-

-

<0.0001>100006.3

0.034

0.00048

2100

-

-

<0.0001>10000

(A73)

9.7

0.029

0.000283600(A36, A73)

2.4

0.036

0.0014710

tRNA (GUA, A73)
tRNA
tRNA
tRNA

Leu
Leu

(GUA)

Lys
Lys

tRNA (G34)
tRNA
tRNA
tRNA

Asp
Asp
Asp

1

_____________________________________________________________________
Numbered nucleotides in parentheses correspond to mutations of tRNAs at numberedpositions. The triplets in parentheses purport the substitutions of ainticodon (Fig. 3).

4. Discussionof the variable arm, straddling both subunits of the

Our present findings together with those of other

TyrRS [18]. Experiments on cross-linking between E.

studies demonstrated that none of the nucleotides incoli tRNAthe acceptor stem, except the discriminator base A73,

that the fragment constituting the variable arm, U46-

are involved in tyrosylation and that the first andG47d, is close to TyrRS in the complex [19]. These

second anticodon nucleotides are required forfindings together with our mutation studies suggest

tyrosylation, while the third is much less important.

that E. coli TyrRS recognizes the long variable arm,

As for the long variable arm, a stem of threebase pairs in length was shown to be important for

Tyrand TyrRS by UV irradiation indicateddirectly binding to the sugar-phosphate backbone in
the vicinity of U46-G47d.

tyrosylation, while the sequence of the arm was not.

The recognition mode of the variable arm in

Various studies on TyrRS based on the crystal

aminoacylation is divergent among class II tRNAs.

structure of Bacillus stearothermophilus TyrRS

For tRNA , both the length and the direction of thesuggest that TyrRS approaches tRNATyr

on the sideSerlong variable arm are crucial for serylation in E. coli

- 202-

Viva Origino 33 (2005) 194 - 207

Thermusdestruction of the overall tertiary structure or change

thermophilus seryl-tRNA synthetase (SerRS) andthe direction of the long variable arm far beyond the

[9,

20].

tRNA

Ser

The

cocrystal

structureofdemonstrated that SerRS binds to the sugar-

differences within class II tRNAs.
The uninvolvement of the third anticodon

phosphate backbone of the long variable arm [21]. In
contrast, the long variable arm of E. coli tRNA

Leu

nucleotide in tyrosylation is intriguing from the view
Tyrdoes not serve as a positive identity determinantpoint of tRNAbecause the primary and secondary structures of thespecific tRNAs, tRNA , tRNA , tRNA , andvariable arm are not correlated to leucylationtRNA , possess the same first and second anticodon

activities [22-23]. These findings imply that leucyl-

nucleotides Q34 and U35, while the third nucleotides

tRNA synthetase (LeuRS) does not recognize thediffer between them; i.e., A36, G36, U36, and C36,

longvariable arm.

For

Tyr

tRNA , thepresent

discrimination. Four amino acidTyr

His

Asn

Asp

respectively. The differences between tRNA

Tyr

and

mutational study showed that the length of the stem

the remaining three tRNAs lie in the long variablein the variable arm, but not its sequence, is required

arm and the discriminator base: C73 for tRNA , andfor tyrosylation. The identity conversion experiments

G73 for tRNA

showed that tRNAtyrosylationSer

activities

and tRNALeu

throughacquired goodchanges

in

the

His

Asp

Asn

and tRNA . Moreover, bothelements are used as the identity determinants ofTyrtRNA . This indicates that tRNATyr

is discriminatedanticodon and the discriminator base alone. This

from other tRNAs containing Q34 and U35 by theindicates that differences in the direction of theexistence of the long variable arm and A73, not by

variable arm within class II tRNAs do not definitely

the third anticodon nucleotide.

affect the recognition of tRNA

Tyr

Among tRNAs containing Q34 and U35,

by TyrRS. This is in

Tyr

His

contrast to the serine system in which the change inneither tRNAthe direction of the variable arm was requisite for the

nucleotide as a major positive identity determinant

identity conversion from tRNA
tRNA

Ser

Tyr

and tRNALeu

to[9, 16]. A previous mutational study showed

nor tRNA

uses the third anticodon[24]. In contrast, C36 of tRNA
tRNA

Asn

Asp

and U36 of

are important for aminoacylation by theirthat the deletion of U47g and U47h at the base of the

cognate aaRS [25, 26]. It is worthy of note thatvariable arm or the insertion of two adenosinestRNA

between C44 and G45 to pair with U47g and U47h

tertiary structure, the long variable arm of tRNA

severely impaired the tyrosylation activity [16].

and the extra G-1 of tRNA , both of which are

These mutations are thought to either cause thepositive identity determinants of the respective

Tyrand tRNA

His

possess the characteristic
Tyr

His

- 203-

Viva Origino 33 (2005) 194 - 207

Trp

tRNAs [27]. Because these tRNAs are easily

variable arm for the discrimination from tRNA . It

discriminated from the other tRNAs by the existence

is more likely that the long variable arm was insertedof the characteristic tertiary structure, they might not

to tRNA

need to use the third anticodon nucleotide as an

the same first and second anticodon nucleotides,

identity determinant.

which already existed in cells. Eukaryotes andNeithertRNA

Tyreukaryotic

norarchaebacterial

possesses the long variable arm [12]. It has

Tyrfor the discrimination from tRNAs witharchaebacteria would have chosen to have C1-G72
instead of the long variable arm.

been reported that C1-G72, G34, Ψ35, and A73 areTyridentity determinants of yeast tRNA , whereas the

Acknowledgments

involvement of A36 in tyrosylation has not been

We are indebted to Professors M. Shimizu, K.

examined yet [28-30]. Taking into account that of all

Nishikawa and T. Yokogawa for their valuable

tRNAs from eukaryotes and archaebacteria only

comments and discussions. We also thank the

Tyr

has C1-G72, thus this unique base pair is

Institute of Space and Astronautical Science and thethought to play a role similar to that of the long

College of Arts and Sciences of the University of

tRNA

Tyr

in discrimination

Tokyo for the use of their facilities. This work wasamong tRNAs with G34Ψ35. We infer that, in

supported by a Grant-in-Aid for the Encouragement

eukaryotes and archaebacteria, A36 is also not

of Young Scientists (to H. A.) (07780549) and a

involved in recognition by TyrRS.

Grant-in-Aid for Scientific Research on Priority

variable arm of eubacterial tRNATyrRS and tryptophanyl-tRNA synthetase(TrpRS) are structural isomers that diverged more
recently

than

most

aaRSs

[31].

evolutionary scenario is that tRNA

Tyr

A

Trp

Education, Science, Sports and Culture of Japan.

proposedacquired thelong variable arm for the discrimination fromtRNA

Areas to (T. H.) (60095023) from the Ministry ofReferences1.

Hasegawa, T. and Himeno, H. The role ofwhen TyrRS and TrpRS were separated andanticodon

UAY and UGG codons were assigned to tyrosine andtryptophan, respectively [32]. However, tRNA

Trp

Tyr

is thought not to need the long

and

the

discriminator

tRNAs by their aminoacyl-tRNA synthetases,
J. Mol. Evol. 35, 436-443 (1992).

identity determinants of tRNA , and vice versa [33].
Tyr

bases

nucleotide in the recognition of some E. coli

possesses C34C35A36 and G73, which are not theThus, tRNA

Shimizu, M., Asahara, H., Tamura, K.,2.

- 204-

Giegé, R., Puglisi, J.D. and Florentz, C.

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8.

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Y.-M. Discriminating

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5035 (1998).

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H., Inokuchi, H. and Nishikawa, K. Co-

and Yokoyama, S. Codon and amino-acidexpression

specificities of a transfer RNA are bothtRNA

converted by a single post-transcriptional

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Escherichia

aminoacylation

14.

Tyr

of

yeast

amber

suppressor

and tyrosyl-tRNA synthetase in

Shimura, Y., Aono, H., Ozeki, H., Sarabhai,

a transfer RNA specificity by removal of

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modified nucleotides, Nature 344, 787-789

tyrosine(1990).

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15.

Nameki, N., Hasegawa, T. and Shimizu, M.

ofaltered

amino

acid

Hou, Y.-M. and Schimmel, P. Modeling within- 205-

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vitro

kinetic

parameters

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the

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16.

elaboration of transfer RNA identity in vivo,

Hasegawa, T., Watanabe, K. and Shimizu, M.

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