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Proc. Natl. Acad. Sci. USAVol. 80, pp. 6755-6759, November
1983Biochemistry
Anticodon loop size and sequence requirements for recognition
offormylmethionine tRNA by methionyl-tRNA synthetase
(synthesis of mutant tRNAs in vitro/RNA-protein
interactions/aminoacylation/T4 RNA ligase)
LADONNE H. SCHULMAN AND HEIKE PELKADepartment of Developmental
Biology and Cancer, Division of Biology, Albert Einstein College of
Medicine, Bronx, NY 10461
Communicated by Harry Eagle, July 21, 1983
ABSTRACT Previous work from our laboratory identifiedseveral
specific sites in Escherichia coli tRNAfMet that are essen-tial for
recognition of this tRNA by E. coli methionyl-tRNA syn-thetase (EC
6.1.1.10). Particularly strong evidence indicated a rolefor the
nucleotide base at the wobble position of the anticodon inthe
discrimination process. To further investigate the
structuralrequirements for recognition in this region, we have
synthesizeda series of tRNAfIet derivatives containing single base
changes ineach position of the anticodon. In addition, derivatives
containingpermuted sequences and larger and smaller anticodon loops
havebeen prepared. The variant tRNAs have been enzymatically
syn-thesized in vitro. The procedure involves excision of the
normalanticodon, CAU, by limited digestion of intact tRNAMet with
pan-creatic RNase. This step also removes two nucleotides from
the3' CpCpA end. T4 RNA ligase is used to join oligonucleotides
ofdefined length and sequence to the 5' half-molecule and
subse-quently to link the 3' and modified 5' fragment to regenerate
theanticodon loop. The final step of the synthesis involves repair
ofthe 3' terminus with tRNA nucleotidyltransferase. The
syntheticderivative containing the anticodon CAU is aminoacylated
withthe same kinetics as intact tRNAfmet. Base substitutions in
thewobble position reduce aminoacylation rates by at least five
or-ders of magnitude. The rates of aminoacylation of
derivativeshaving base substitutions in the other two positions of
the anti-codon are 1/55 to 1/18,500 times normal. Nucleotides that
havespecific functional groups in common with the normal
anticodonbases are better tolerated at each of these positions than
thosethat do not. A tRNAfMet variant having a six-membered loop
con-taining only the CA sequence of the anticodon is
aminoacylatedstill more slowly, and a derivative containing a
five-membered loopis not measurably active. The normal loop size
can be increasedby one nucleotide with a relatively small effect on
the rate of ami-noacylation, indicating that the spatial
arrangement of the nu-cleotides is less critical than their
chemical nature. We concludefrom these data that recognition of
tRNAf¶et requires highly spe-cific interactions of methionyl-tRNA
synthetase with functionalgroups on the nucleotide bases of the
anticodon sequence.
We have previously studied the effect of chemical modifica-tions
at 25 different sites in Escherichia coli tRNAfMet on theability of
the tRNA to be aminoacylated by E. coli methionyl-tRNA synthetase
(EC 6.1.1.10) (1, 2). Most of these structuralalterations did not
significantly impair the interaction of tRNAfmetwith Met-tRNA
synthetase; however, modification of specificnucleotides in three
structural regions drastically reduced me-thionine acceptance.
These results focused our attention on theanticodon, the variable
loop, and the acceptor stem of tRNAfMetfor more detailed analysis
of the structural requirements forprotein-tRNA recognition. We have
shown that the anticodonwobble base plays an essential role in this
process (3, 4). In this
paper, we describe the results of a systematic examination ofthe
effects of alterations in anticodon loop size and sequenceon
recognition of tRNAMet by Met-tRNA synthetase.
MATERIALS AND METHODSMaterials. Nucleoside 3'-phosphates,
nucleoside 5'-diphos-
phates, nucleoside 3',5'-bisphosphates, poly(A,C), and GpApCwere
purchased from P-L Biochemicals. Nucleoside 5'-mono-phosphates,
dinucleoside monophosphates, GpCpC, and GpCpUwere obtained from
Sigma. [y-32P]ATP, [a-32P]ATP, and [ S]-methionine were purchased
from Amersham. E. coli tRNAfmet(1.8 nmol/A260 unit),
primer-dependent Micrococcus luteuspolynucleotide phosphorylase,
calf intestinal alkaline phospha-tase, and nuclease P1 were
obtained from Boehringer Mann-heim. RNases T1 and U2 were purchased
from Calbiochem andPhy M and Bacillus cereus RNases were from P-L
Biochemi-cals. E. coli Met-tRNA synthetase was purified from E.
coli K-12 strain EM 20031 (5) and T4 RNA ligase was purified fromE.
coli infected with T4 phage strain SP62, amN82 (6) as de-scribed.
Purified rabbit liver tRNA nucleotidyltransferase wasa gift from M.
Deutscher.
Synthesis of Oligonucleotides. The trinucleotides GpCpA andCpApU
were synthesized by reaction of GpC with ADP andCpA with UDP, using
polynucleotide phosphorylase as de-scribed by Thach and Doty (7).
CpApGp was synthesized by asimilar reaction of CpA with GDP in the
presence of RNase T1at 250 units/ml. CpAp was obtained from a
digest (18 hr at37°C) of poly(A,C) (1:1) with RNase U2 (0.5 unit/mg
of RNA)in 50 mM sodium acetate, pH 4.5, followed by incubation
with0.125 M HCI at room temperature for 6 hr. The tetranucleo-tides
GpCpApCp and GpCpUpAp were synthesized by addi-tion of pCp to GpCpA
and pAp to GpCpU, using T4 RNA ligase(8). Treatment of the
tetranucleotides with RNase T1 yieldedthe trinucleotides CpApCp and
CpUpAp. The trinucleotidesCpApUp, CpCpUp, ApCpUp, and CpUpUp were
similarlysynthesized by ligase-catalyzed addition of pUp to the
corre-sponding trinucleoside diphosphates followed by cleavage
ofthe resulting tetranucleotides with RNase T1. The
tetranucleo-tide CpApUpAp was synthesized by addition of pAp to
CpApU,using RNA ligase. All oligonucleotides were purified by
columnchromatography and analyzed as described elsewhere (3).
Oli-gonucleotides were phosphorylated at the 5' terminus by
using[y-32P]ATP and PseT 1 polynucleotide kinase (9).
Synthesis of tRNAflet Containing Altered Anticodon
LoopSequences. Half-molecule-sized fragments of tRNAM&et
miss-ing the anticodon nucleotides and two nucleotides of the 3'
ter-
Abbreviations: tRNATA'ut, tRNA containing the sequence CAU in
theanticodon position that has been enzymatically synthesized in
vitro fromhalf-molecule-sized fragments of Escherichia coli tRNA
Met (other syn-thetic tRNAs are similarly indicated by the
sequences in their anticodonloops); p*, 32P-labeled phosphate.
6755
The publication costs of this article were defrayed in part by
page chargepayment. This article must therefore be hereby marked
"advertise-ment" in accordance with 18 U.S.C. §1734 solely to
indicate this fact.
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6756 Biochemistry: Schulman and Pelka
minal CpCpA sequence were isolated after limited digestion ofthe
tRNA with pancreatic RNase (3). Intact molecules contain-ing normal
and altered anticodon loop sequences were synthe-sized by using RNA
ligase and polynucleotide kinase,_ and the3'-terminal sequence was
enzymatically replaced by using tRNAnucleotidyltransferase, as
described before (3).
Aminoacylation of Modified tRNAfMet. Reaction mixturesfor assay
of methionine acceptance (0.05-0.15 ml) contained0.01-0.2 ,uM tRNA,
20 mM imidazole HCI at pH 7.5, 0.1 mMEDTA, 2 mM ATP, 4mM Mg9l2,
150mM NH4Cl, bovine serumalbumin at 10 Ag/ml, and 17AM
[3S]methionine (1,500-100,000cpm/pmol). Samples were equilibrated
at 260C for 5 min andreactions were initiated by addition of
purified E. coli Met-tRNAsynthetase. Incubation at 26°C was
continued for various timesand aliquots (5-45 ,ul) were pipetted
onto 2.5-cm Whatman 3MM filter disks, and the disks were added to
cold 10% tri-chloroacetic acid containing I mM unlabeled methionine
(10 mlper filter) and washed for 10 min;followed by four washes
withcold 5% trichloroacetic acid and one wash with 95%
(vol/vol)ethanol. The filters were dried for 20 min under a heat
lamp
.and-placed in scintillation vials. with 5 ml of Econofluor
(NewEngland Nuclear), and the radioactivities were measured in
aliquid scintillation counter. Aliquots were taken in triplicate
formeasurement of aminoacylation yields in 30-min incubations
at26°C at various enzyme concentrations. Initial rates of
ami-noacylation were measured under conditions iii
which-methi-onine incorporation was linear with time and
proportional toenzyme concentration. Aminoacylation kinetics were
measuredat 0.04 nM enzyme for tRNAfMet and tRNACm' (aliquots
takenat 1-min intervals), 0.4 nM for tRNAmtA (aliquots taken at
0.5-min intervals), 4 nM for tRNAfcmIu (aliquots taken at
0.5-minintervals), 40 nM for tRNAfmueu, tRNAftGt, and tRNAAJueA
(ali-quots taken at 1-min intervals), and 40 nM for tRNOAect
andtRNAfMAet (aliquots taken at 5-min intervals). Initial rates
weremeasured at five different tRNA concentrations except for
theCUA derivative (two). Relative initial rates of
aminoacylationwere determined after calculation of the moles of
methionineincorporated per mole of enzyme per minute for each
tRNAderivative. Reaction mixtures containing enzyme but no tRNAor
enzyme plus equivalent amounts of yeast tRNAPhe gave thesame blank
values for methionine acceptance and were pre-pared in parallel
with each assay. Derivatives that were inactiveat low Met-tRNA
synthetase concentrations were tested for theirability to inhibit
aminoacylation of tRNAfmet in reaction mix-tures (50 ,ul) that
contained 0.03 MM tRNA et, 0.06-0.3 MtRNAfMet derivative, and 40pM
enzyme. Incubations were at26°C and 9-,u1 aliquots were withdrawn
at 1-min intervals formeasurement of methionine acceptance.
Parallel incubation mix-tures containing enzyme but no tRNA or
enzyme plus inhib-itor but no unmodified tRNAfMet gave the same
blank values.
RESULTSSynthesis and Structural Characterization of tRNAMet
De-
rivatives. Derivatives of tRNAfet containing alterations in
an-ticodon loop size and sequence were synthesized by a proce-dure
analogous to that described previously (3). Limited digestionof
tRNAfmet with pancreatic RNase was used to generate
half-molecule-sized fragments missing the anticodon nucleotides
andthe two terminal nucleotides of the 3' CpCpA sequence.
Thedephosphorylated 5' half-molecule was annealed with the
3'half-molecule -containing a3'-phosphate group.
5'-32P-Labeledoligonucleotides of different length and sequence
were joinedto the 3'-OH group of the 5' fragmentby using T4 RNA
ligase.The extended 5' fragment and the 3' fragment were
dephos-phorylated at the 3' termini-and phosphorylated at the 5'
ter-mini with polynucleotide kinase and [y-32P]ATP. The
anticodon
loop was joined by incubation of the annealed complex withRNA
ligase and the 3'-terminal CpCpA sequence was enzy-matically
repaired by using tRNA nucleotidyltransferase in thepresence of
unlabeled CTE and [a-32P]ATP. The final productswere isolated by
polyacrylamide gel electrophoresis in 7 M urea.All of the tRNA
derivatives migrated in the position expectedon the basis of the
length of the oligonucleotide inserted in theanticodon loop except
for tRNAMAeGt. Several different prepa-rations of this derivative
migrated more slowly on denaturinggels than the products prepared
by other trinucleotide inser-tions. Low specific activity 32p
labels were incorporated at the5' and 3' termini and at the sites
of joining in the anticodonloop, facilitating calculation of yields
and product purity at eachstage of the synthesis. Results were
similar to those obtainedpreviously for synthesis of tRNA!let
derivatives containing basesubstitutions in the wobble position
(3). Anticodon loop closureand CpCpA repair were essentially
quantitative in all cases. Theoverall recovery of the desired tRNA
derivatives was limited bythe incomplete addition of
oligonucleotides to the 5' half-mol-ecule. The desired adducts were
obtained in yields of 15-25%;however, partial degradation of the
initial products by reversereactions of RNA ligase (10)
necessitated fractionation of thereaction mixtures on denaturing
polyacrylamide gels and re-duced the yield of desired product by as
much as 50% in somecases.
Each tRNA derivative was digested with T1 RNase and
theresulting'32P-labeled oligonucleotides were separated by
chro-matography on DEAE-ceHuloserin the presence of 7 M urea.All
samples yielded the expected 5' and 3' oligonucleotidesP*CpGp and
CpApApCpCp*A plus an additional oligonucleo-tide derived from the
anticodon loop region. The size of thisoligonucleotide was
determined by chromatography with un-labeled oligonucleotide
markers. The anticodon sequence ofnormal tRNAfmet is found in an
11-residue oligonucleotide,CmpUpCpApUpApApCpCpCpG, after digestion
with T1 RNase.The corresponding synthetic derivative yielded the
labeledproduct CmpUp*CpApUp*ApApCpCpCpG, which migratedwith the
unlabeled oligonucleotide marker and yielded CmpUp*and ApUp* in a
1:1 ratio after further digestion with pancreaticRNase. Derivatives
synthesized by insertion of CCU, CUU, ACU,CUA, and CAC in the
anticodon loop also gave 32P-labeled un-decanucleotides on
digestion with T1 RNase, while insertion ofCAUA and CACA yielded
dodecanucleotides, insertion of CAyielded a decanucleotide, and
insertion of C yielded a nonanu-cleotide. In each case, further
digestion of the isolated oligo-nucleotide with pancreatic RNase
gave the expected 32P-la-beled products. The tRNAfMet derivative
synthesized by insertionofCAG yielded the T1 RNase product
CmpUp*CpApGp*, whichgave CrpUp* and ApGp* in a 0.9:1.0 ratio after
digestion withpancreatic RNase. On the basis of incorporated
radioactivity,the products are 70-90% pure. On the basis of the
ratio of 32Pto A2w, specific activities of 800-1,000 pmol/A2m unit
are ob-tained. As noted previously (3), similar specific activities
areobtained for equivalent amounts of intact tRNAfmet after
elec-trophoresis and isolation from polyacrylamide gels due to
con-tamination of small samples with nondialyzable,
ethanol-pre-cipitable UV-absorbing material from the gels. The 32p
labelincorporated during the synthesis has therefore been used
tocalculate the actual concentration- of each product for
amino-acylation studies.
As a further check on the synthetic procedure, tRNA deriv-atives
were labeled at the 5' termini. by using polynucleotidekinase and
high specific activity [y-32P]ATP for gel sequenceanalysis by the-
method of Donis-Keller et al. (11). Sample au-toradiograms obtained
from sequencing the products synthe-sized by insertion of the
trinucleotides CAU, CAG, and ACU
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Proc. Natl. Acad. Sci. USA 80 (1983) 6757
tRNA fMetRNCAU tRNAMetRNCAG tRNAACUG A A+U U+C G A A+UU+C G A
A+U U+C
umIO G70G64 G__063 0700
___ iG64G53 loGom G53
G5i2 _ 553 ^52 _ _49 _ G524of '1-49
045 _- _
04242* G175 _({042G42
.~JS3.G 32'G29
!L,7
G31030G29
G26G26
[AI--S LeviG3iG30
_ G29
_ G26
FIG. 1. RNA sequencing gels of 5'-32P-labeled tRNAft
deriva-tives. Lanes G, partial RNase T1 digest; lanes A, partial
RNase U2 di-gest; lanes A+U, partial RNase Phy M digest; lanes U+C,
partial B.cereus RNase digest. The region of each sequence
corresponding to theanticodon nucleotides is enclosed in a box. The
numbering of nucleo-tides in tRNAfht is based on the system adopted
in ref. 12. The bandcorresponding to C35 in tRNAfc§ is fainit due
to much more rapidcleavage at U36.
in the anticodon loop are shown in Fig. 1. The sequencing
gelsconfirm that the structure of each synthetic tRNA
derivativecorresponds to the predicted structure. All of the data
indicatethat the CAG derivative has the same sequence as
tRNAMetexcept for the U--G base substitution in the anticodon,
andwe, therefore, conclude that the abnormal migration of this
de-rivative on polyacrylamide gels is due. to an altered
confor-mation under the partial denaturing conditions of the
electro-phoresis.
Aminoacylation of tRNAfmet Derivatives. The kinetic pa-rameters
for aminoacylation of tRNATAU were compared withthose obtained with
intact tRNAfmet and a control sample thatwas isolated from a
denaturing polyacrylamide gel in parallelwith the synthesized
tRNAs. Intact tRNAfmet was aminoacyl-ated with a Km of 0.8 A.M and
a VmX, of 3 jumol/min per mg.Different preparations of control
tRNANet isolated from thegel and tRNAAgu had the same Km but a
20-40% lower Vm,.We conclude that a contaminant derived from the
gel has a smalleffect on the rate of aminoacylationlby Met-tRNA
synthetase;however, different preparations of the synthesized tRNAs
gavevery similar results, indicating that the effect is relatively
con-stant for different samples.
Aminoacylation of the synthesized tRNA derivatives con-taining
anticodon loop modifications was examined at a varietyof Met-tRNA
synthetase concentrations (Fig. 2). Under con-
Table 1. Aminoacylation of tRNAfmet derivatives containing
basesubstitutions in the anticodon
Synthesized Moles of methionine acceptedanticodon per mole of
tRNA in 30 mintsequence* 0.4 nM 4 nM 40 nMCAUCAUA
(+1)CCUCUUCUACAGCACCA(-1)CACA (+1)C(-2)ACU
1.00.320.130.01
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6758 Biochemistry: Schulman and Pelka
Table 2. Initial rates of aminoacylation of tRNAfMt
derivativesat 40 nM tRNA
tRNA*tRNAfMettRNAfMet
(gel)tCAUCAUACCUCUUCUACAGCACCACACUUAUAAUGAU
Moles Met-tRNA permole Met-tRNA
synthetase per min28.4522.8022.151.59
4.0 x 10-12.6 x 10-22.0 x 10-21.7 x 10-21.2 x 10-30.5 x
10-3106
* The oligonucleotide inserted in the anticodon loop of
synthesizedtRNAfMet derivatives is indicated.
t Control sample isolated from a denaturing polyacrylamide gel
in par-allel with the synthesized tRNAfmet derivatives.
t Only one sample of this derivative has been assayed.
ative order of aminoacylation activity as seen for the
purifiedtRNAfmet derivatives was found. Heat treatment of
isolatedproducts in the presence of Mg2" under conditions used to
re-nature other tRNAs also failed to increase the levels of
me-thionine acceptor activity.The apparent rates of aminoacylation
shown in Table 2 have
not been corrected for any effect of anticodon base
substitu-tions on the rate of enzymatic deacylation by Met-tRNA
syn-thetase. Alterations in the relative rates of the forward and
re-verse reactions catalyzed by aminoacyl-tRNA synthetases
arenormally characterized by observation of a low plateau level
ofproduct, the final value of which is dependent on the
initialenzyme concentration (13). Aminoacylation of the
anticodon-substituted tRNAfmet derivatives does not reach a
prematureplateau value, however, but continues to show a slow
increasein the amount of product formed with time in prolonged
in-cubations (Fig. 2). This suggests that the major effect of
thesesubstitutions is to reduce the rate of the forward reaction
ratherthan to increase the rate of enzymatic deacylation. The
relativerates shown in Table 2 are therefore believed to accurately
re-flect the relative effects of alterations at different positions
ofthe anticodon on aminoacylation, although the absolute
forwardrates may be underestimated.The most defective tRNA
derivatives were examined for their
ability to compete with normal tRNAfmet during aminoacyla-tion
under conditions of limiting enzyme concentration. A 10-fold excess
of wobble base substituted tRNAs, tRNAfm'! ortRNAfCMet, or a 5-fold
excess of tRNAfCAkC or tRNAfAG had nodetectable effect on the rate
of aminoacylation of control tRNAsamples. Due to a shortage of the
synthesized tRNAs, higherconcentrations were not tested.
DISCUSSION
The data presented in this paper and in previous publications(3,
4) indicate a crucial role for the anticodon nucleotides oftRNAelet
for recognition by Met-tRNA synthetase. Aminoacyl-ation is reduced
to below levels of experimental detection bybase substitutions in
the wobble position, and lesser, althoughstill dramatic, effects
result from structural changes at the other
two positions of the anticodon. Base substitutions could
alterinteraction of tRNAfmet with Met-tRNA synthetase by
elimi-nation of essential ligands, by steric or electrostatic
interfer-ence with binding, or by introducing conformational
changesthat alter access of the enzyme to its normal binding sites
onthe tRNA. While we cannot entirely exclude the possibility
thatthe synthesized tRNA derivatives have a conformation
signif-icantly different from that of native tRNA let, this seems
anunlikely explanation for the wide range of effects of
anticodonbase substitutions on aminoacylation rates. In addition,
we havepreviously examined the conformation of a similar
tRNAfmetderivative containing uridine in the wobble position by
high-resolution NMR spectroscopy. Chemical deamination of thewobble
cytidine to uridine eliminated methionine acceptor ac-tivity but
produced no detectable loss of the secondary or ter-tiary N.H
hydrogen bonds found in the native structure (2).The available data
also indicate that small changes in the ge-
ometry of the anticodon loop, such as those resulting from
py-rimidine-purine substitutions or changes in the inherent
basestacking properties of different nucleotides, cannot account
forthe large effects of anticodon base substitutions on
aminoacyl-ation rates. Enlargement of the anticodon loop by one
nucleo-tide, which would be expected to cause a similar small shift
inthe position of anticodon nucleotides, has a relatively small
ef-fect on the aminoacylation rate. On the other hand,
tRNAfmethaving the permuted anticodon sequence ACU, with a
normalpurine-to-pyrimidine ratio and anticodon loop size, has no
de-tectable activity. These results indicate that while the
spatialarrangement of the loop plays some role, the major factor
inrecognition of tRNAfmet by Met-tRNA synthetase involves
in-teraction of the enzyme with specific functional groups on
an-ticodon nucleotides. The most stringent requirement is for
acytidine base in the wobble position. The inability of other
basesto substitute even weakly for cytidine suggests that there
maybe a more extensive interaction of the enzyme with this
nu-cleotide than with other anticodon bases, possibly involving
si-multaneous binding to the ribophosphate backbone and to
func-tional groups on the pyrimidine ring.The magnitude of the
effect of sequence changes at the oth-
er two positions of the anticodon depends on the structure ofthe
substituted base, those nucleotides having specific function-al
groups in common with the normal nucleotides being tolerat-ed
better than those that do not. Thus, cytidine inserted inthe middle
position of the anticodon contains the structure-N=C(NH2)-, found
at positions 1 and 6 of the normaladenosine nucleotide, and yields
a tRNAfmet derivative that isamino-acylated at a rate 15-fold
higher than that of the cor-responding uridine derivative lacking
the basic ring nitrogenand exocyclic amino group. Similarly,
guanosine contains the-NH-CO- structure found at the 3 and 4
positions of theuridine ring and is a 14-fold more effective
substitute for uri-dine at the 3' end of the anticodon than is
cytidine. The greaterthan 104-fold reduction in the rate of
aminoacylation of thetRNAf9A'c derivative suggests that there may
be a negative ef-fect of cytidine at this site in addition to the
loss of some pos-itive interaction(s) with the normal uridine
nucleotide.Few quantitative data on the relationship between
anticodon
sequences and aminoacylation activity are available for
othertRNAs. Bruce and Uhlenbeck have carried out a systematic
studyof the effects of base substitutions at each position of the
an-ticodon on aminoacylation of yeast tRNAPhe (14, 15). A
10-foldincrease in Km resulted from alterations in the wobble base,
andsmaller effects were produced by changes at other
anticodonpositions. These results indicate that no specific
anticodon baseis essential for recognition of tRNAPhe by its
cognate aminoacyl-tRNA synthetase; however, there may be a
cumulative effect
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on binding of interaction of Phe-tRNA synthetase with a num-ber
of sites in this and other regions. On the other hand, largeeffects
on aminoacylation rates have been shown to result fromsingle base
changes in the anticodon sequences of beef tRNATrP,yeast tRNAVaI,
E. coli tRNAGIY, and E. coli tRNA"'9 (16-19),and a C-to-U change in
the middle position of the anticodon ofE. coli tRNATrP increases
the rate of mischarging of this tRNAwith glutamine by five to six
orders of magnitude (20). The ef-fect of the same anticodon
alteration on aminoacylation oftRNATrP by the cognate Trp-tRNA
synthetase is significant butmuch smaller, a 59-fold increase in Km
and 6.7-fold reductionin Vmax being observed (20). The available
data therefore in-dicate a wide range of effects of anticodon base
changes on ratesof acylation and misacylation by aminoacyl-tRNA
synthetases,with E. coli Met-tRNA synthetase showing stringent
structuralrequirements for recognition in this region. The lack of
a cy-tidine base in the wobble position of a noncognate tRNA
wouldbe sufficient to exclude its aminoacylation by Met-tRNA
syn-thetase. Thus, it is possible that only a small number of
otherunique sites are required to ensure complete accuracy in
sub-strate selection by this enzyme. Chemical modification and
en-zymatic excision data (1) have revealed that alterations in
manydifferent regions of the structure of tRNAfmet have little or
noeffect on methionine acceptor activity; however, modificationof a
specific G residue in the variable loop or on the 3' side ofthe
acceptor stem drastically reduces the rate of aminoacylation(21,
22). Small changes in Km or Vm., also result from
structuralalterations at other specific sites in the acceptor stem
region (1,2, 23).
The mechanism by which wobble base substitutions
reduceaminoacylation rates of tRNAfmet by five or six orders of
mag-nitude remains unclear. It seems improbable that the
smallnumber of hydrogen bonds that could uniquely be made
withcytidine could provide a sufficient difference in binding to
ac-count for the observed rate differences or that interference
byother nucleotides in the wobble position could be sufficient
toexclude binding. It is more plausible that interaction of
Met-tRNA synthetase with this cytidine leads to a rearrangement
ofthe complex that increases the number of binding contacts ina
cooperative fashion or that induces a long-range conforma-tional
change that greatly enhances the rate of the catalytic
step.Evidence for such an anticodon-dependent rearrangement hasbeen
reported for other tRNA-synthetase complexes. Excisionof the Y base
from the anticodon loop of yeast tRNA"he has beenshown to result in
an altered interaction of the 3' terminus ofthe tRNA with Phe-tRNA
synthetase (24), and tRNAT`P has beenshown to induce a
conformational change in beef Trp-tRNAsynthetase that is dependent
on the sequence of the anticodonloop (25).
The structural requirements for interaction of
Met-tRNAsynthetase with the anticodon bases of its tRNA substrates
ap-pear to involve highly localized sites on each nucleotide
base,because the enzyme is relatively unaffected by
significantstructural alterations only a few atoms removed from the
ap-parent contact sites. For example, saturation of the 5,6
doublebond of the uridine at the 3' end of the anticodon with
bisulfite
ion has only a small effect on the rate of aminoacylation
oftRNAfmet (5) and the presence or absence of an acetyl moietyon
the exocyclic amino group of the wobble base has no effecton the
interaction of Met-tRNA synthetase with tRNAmet (26).Construction
of tRNA derivatives containing base analogs moreclosely related to
the natural nucleotide should assist in dif-ferentiating between
positive and negative interactions of Met-tRNA synthetase with
specific bases at each site of the anti-codon and allow further
resolution of the number and locationof essential functional groups
in this region.
This work was supported by American Cancer Society Grant
NP-19.Partial salary support for L. H.S. was provided by National
Cancer In-stitute Grant P30 CA1333 0-10.
1. Schulman, L. H. & Pelka, H. (1977) Biochemistry 16,
42564265.2. Schulman, L. H. (1979) in Transfer RNA: Structure,
Properties
and Recognition, eds. Schimmel, P. R., S611, D. & Abelson,
J. N.(Cold Spring Harbor Laboratory, Cold Spring Harbor, NY),
pp.311-324.
3. Schulman, L. H., Pelka, H. & Susani, M. (1983) Nucleic
Acids Res.11, 1439-1455.
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