Mutant enzymes and tRNAs as probes of the glutaminyl-tRNA synthetase: tRNAGln interaction

Biochimie ( 1991 ) 73, 1501-1508 © Socirt6 fran~aise de biochimie et biologic mol6culaire / Elsevier, Paris

150 I

Mutant e n z y m e s and t R N A s as probes of the glutaminyl-tRNA synthetase: t R N A GIn interaction S Englisch-Peters**, J Conley, J Plumbridge***, C Leptak, D $611", MJ Rogers Department of Molecular Biophysics and Biochemistry, Yale Universi~. , New Haven, CT 0651 I, USA (Received 1 September 1991; accepted 10 October 1991 )

Summary - - This paper focuses on several aspects of the specificity of mutants of Escherichia coli glutaminyl-tRNA synthetase (GInRS) and tRNAGIn. Temperature-sensitive mutants located in ginS, the gene for GInRS, have been described previously. The mutations responsible for the temperature-sensitive phenotype were analyzed, and pseudorevertants of these mutants isolated and characterized. The nature of these mutations is discussed in terms of their location in the three-dimensional structure of the tRNAC~n:GInRS complex. In order to characterize the specificity of the aminoaeylation reaction, mutant tRNAGIn species were synthesized with either a 2'-deoxy AMP or 3'-deoxy AMP as their 3'-terminal nucleotide. Subsequent assays for aminoacylation and ATP/PPI exchange activity established the esterification of glutamine to the 2'-hydroxyl of the terminal adenosine; there is no glutaminylation of the 3'-OH group. This correlates with the classification of GInRS as a class I aminoacyl-tRNA synthetase. Mutations in tRNAoln are discLssed which affect the recognition of GlnRS and the current concept of glutamine identity in E coli is reviewed. aminoacyl-tRNA synthetase / tRNA / mutants / glutamine

Introduction A m i n o a c y l - t R N A synthetases catalyze the esterification of amino acids to the 3'-end o f t R N A (reviewed in [1]). Precision in this step is essential to the accuracy of gene expression. The E coil G I n R S has been the focus of study for m a n y years, and is IJI O O d O l y

UIIK;

UI

t l IK:;

Ut:;bl.

LI! I U ~ ; I ~ L U U U

~,y 1 ILl ll~;tdbK,~t

c o n c e r u m g the specificity of t R N A recognition. Mutations which lower the specificity o f GInRS have been isolated by mischarging o f supF, the a m b e r suppressor derived from tRNATy r ([2]; s u m m a r i z e d in [3]). This misacylation was tested in vivo f r o m the suppression o f the laCZlooo mutation. The mischarging capacity was subsequently shown to be an inherent characteristic of GInRS and the ratio o f these substrates in vivo is critical in maintaining the accuracy o f aminoacylation. W h e n the ratio o f G I n R S to t R N A Cln in the cell is elevated, GInRS will m i s a m i n o acylate supF t R N A ~ r with glutamine [41. This mischarging in vivo by overproduction o f G I n R S is abolished by concomitant overproduction o f t R N A G~" [4]. *Correspondence and reprints **Present address: Department of Biochemistry, Glaxo SpA, via Fleming 2, 1-37100 Verona, Italy; ***Present address: Institut de Biologic Physico-Cbimique, 13, rue Pierre et Marie Curie, F-75005, Paris, France

The lacZ~ooo mutation is extremely useful to test for charging by GInRS in vivo, as only an a m b e r suppressor t R N A inserting glutamine at the site of the mutation will give a Lac ÷ phenotype. We have made use o f this powerful genetic selection for mutants of GInRS that confer mischarging o f supF [21 and more recently a mutant of the a m b e r suppressor derived from tRNA set was made that is recognized by GinRS ([5]; s u m m a r i z e d in [31). The cloning and overexpression of ginS, the gene for GInRS, and the glnV genes for the tandemly duplicated t R N A , Gt" isoacceptor, has led to the crystallization o f the tI~NAGIn:GinRS complex with ATP [6]. The structure o f the complex at 2.8 A resolution was then determined by X - r a y crystallography [7], and further refined at 2.5 A resolution [8].

Temperature-sensitive mutants of

glnS

Characterization of temperature-sensitive mutants and pseudorevertant temperature-resistant mutants The temperature-sensitive mutation ginS1 was isolated and characterized a n u m b e r o f years ago [91. The mutation was generated by EMS mutagenesis, and genetic m a p p i n g established that glnSl is located in the structural gene fo- GInRS. Complementation o f the temperature-sensitive mutation enabled cloning o f

S Englisch-Peterset al

! 502

the ginS gene for wild-type GInRS [10] and also established that mutations conferring mischarging of supF tRNA%, are located in the gene for gb~.S [2]. A second mutation ginS172 was isolated from a procedure for isolating spontaneous temperaturesensitive mutants for RNA and/or protein [11]. By subcloning both the ginS1 and ginS172 genes from the mutant E coil strains the mutations were mapped to the same 948-bp BgllI fragment within the structural gene for ginS [12, 13]. Subsequent DNA sequence analysis has established the corresponding amino acid changes in GlnRS responsible for the temperaturesensitive phenotype. The glnS1 mutation (G to A) replaces Glu in position 222 with Lys in the GInRS 1 gene product, The mutation glnS172 was identified as an A to C change replacing Thr in position 266 with Pro in GInRSI72 (summarized in fig 1). The mutated GlnRS 1 protein was shown in vitro to have an altered K~ for tRNA GIn, and overproduction in vivo of tRNA C~n appears to partially complement the ginS1 strain; no complementation is seen with the ginS172 strain [9, 14]. Moreover, thermo-resistant second site revertants of ginS1 have also been isolated that increase the level of tRNA GIn as well as several other tRNA species [ 15, 16]. Therefore, excess tRNA can alleviate the temperature-sensitive phenotype of the mutated GInRS enzyme, analogous to the cloned tRNA eh~ complementing an E coli PheRS temperature-sensitive mutation [17]. Since GInRS1 is unaltered in the affinity for the amino acid [9], it is then possible that this temperature-sensitive mutant is altered in tRNA recognition. In an attempt to learn more about both mutation sites, spontaneous temperature-resistant revertants were isolated. Individual colonies were grown to satumtlnn ~t "~noc',~ ~.~'~a ,h, m.,,~ v"''~'1~*~'* at -,-,An°c',..ov~lm/~m.:~"" Cultures of individual isolates from these revertants were grown and examined for thermostability. A number of the thermo-resistant mutants obtained from glnSl and glnS172 were cloned and sequenced. Of

Gene

Protein

ginS +

GInRS

I__E222

ghlSl

E222K

!

K222Q

I - - Q2,_2----553

T266P P266S P266L

1

ginS172

Phenotype

i

l

T266~553

K222___553

-P266 -S266 L266

553 55S 553

TR ts

TR tS TR TR

Fig 1. The corresponding amino acid changes in the temperature-sensitive (ts) mutants of GInRS, and the changes in the pseudorevertant temperature-resistant (TR) mutants of the ginS gene. The one-letter amino acid abbreviation is used in the changes from the wild type (gins+) gene.

these, some were true revertants and now possessed the wild-type sequence in the ginS gene. Other isolates were pseudorevertants; one isolate led to a K222Q change at the site of the ginS1 mutation, while pseudorevertants of glnS172 gave rise to P266S and P266L (fig 1). The thermostabiiity of both temperature-sensitive and temperature-resistant mutant enzymes was examined in vitro. An equivalent amount of a crude GInRS preparation from each extract was incubated for various lengths of time at 43°C, and then assayed for aminoacylation activity for 15 min at 32°C to estimate the percentage of GInRS activity remaining. The thermo-resistant enzymes P266S and P266L (derived from glnS172) showed comparable activity to the wild-type enzyme when assayed in vitro at 30°C. However, after a 15-min incubation at 43°C, there was a 50% drop in activity of the wild-type enzyme while P266S and P266L showed a 70% decrease. For comparison, no activity of the thermosensitive enzymes GlnRS1 and GlnRS172 is detected after a 5min incubation at 43°C. The thermo-resistant pseudorevertant derived from GInRS1 (K222Q) exhibited only about one-fifth the activity of the other pseudorevertants in vitro at 30°C. Incubation at 43°C for 15 min resulted in about 50% of residual activity.

The temperature-sensitive mutations are not located near" substrate binding sites In the structure of the GInRS protein complexed with tRNA ctn the amino terminal portion of the enzyme consists of a conserved structural motif, the dinucieotide or Rossmann fold, that binds ATE In the GInRS:tRNAGIn structure this motif is interrupted by a domain that binds the acceptor stem of tRNA [7]. The temperature-sensitive mutants and pseudorevertants of glnSl and glnS172 are altered at amino acids 222 and 266 in the respective enzymes (fig 1). These positions are both within the second half of the dinucleotide fold, and E222 in the wild-type enzyme makes strong polar interactions with residues of the first part of the dinucleotide binding domain and with a residue in the acceptor binding domain [18]. The mutation in GInRS1 (E222K) may decrease the thermal stability of the enzyme by steric or electrostatic effects of the mutation, which could be alleviated by the pseudorevertant K222Q. The amino acid T266, mutated to P in GInRS172, is an interior residue and this mutation may destabilize the peptide backbone conformation in the temperature-sensitive enzyme. The greater thermostability conferred by the conservative substitution T266S implies that the hydroxyl group is of some importance in maintaining protein stability. On the other hand, the lesser stability of the T266L may be due to the inability of the bulkier Leu to restore the tight packing of the backbone in this region.

! 503

tRNA °in interac!ion Clearly, the positions of the amino acids altered in the temperature-sensitive enzymes do not directly interact with the substrates. The observed increase in KM for tRNA al. with GlnRS I [9] may then be a result o f the long-range conformational alteration of the e n z y m e to affect tRNA binding as a consequence o f altered protein stability, resulting in protection o f the mutated enzyme by excess tRNA C]n. The mutated G I n R S I 7 2 enzyme, w *h the change at amino acid 266 which is buried more deeply within the protein ]7], is then less likely to have the phenotype reverted by overproduction of tRNA tin. Further studies are in progress to define in greater detail the molecular parameters for the accurate recognition o f tRNA. The mutants of GInRS obtained by genetic selection based on mischarging o f supF ([2]; summarized in [3]) were subsequently shown from the structure of the wildtype enzyme to interact with the acceptor stem o f tRNA GIn [19]. ~ direct interaction with Asp235 (which is altered in the mischarging mutant enzymes GInRS7 and GInRS10 [19]) and nucleotide G3 therefore implicates this interaction as important for tRNA discrimination. In the temperature-sensitive GInRS enzymes, no direct interaction with the substrates is likely to be affected by the mutation to the temperature-sensitive proteins.

The 2'-hydroxyl-group of E coil tRNA Gin is the site of aminoacylation The question o f which hydroxyl group (2' or 3') o f the terminal adenosine is aminoacylated was addressed more than a decade ago. The results were used to divide the synthetases into 3 classes o f enzymes; those •

.a.,

.,,,.#Ll,

~ll

t.l|¢,...

Az

or at both hydroxyl groups (summarized in [20]). The yeast GlnRS enzyme was placed in the 3'-OH class [21] but the E coli e n z y m e was not included in this survey. However, the yeast and E coil enzymes show a high degree o f homology, apart from a long Nterminal extension in the yeast e n z y m e [22]. It is then interesting to see if the site o f aminoacylation is a conserved feature of the GInRS enzymes. The more recent classification o f aminoacyl-tRNA synthetases into class I and class II enzymes [23, 24] was based on limited sequence and structural homology. Strikingly, there is a strong correlation between class I synthetases and acylation o f the amino acid to the 2'O H o f the terminal adenosine. There is good structural homology o f GInRS to other class I synthetases in the dinucleotide fold as well as in other parts o f the enzyrne [25]. By these criteria it is expected that GlnRS should catalyze the esterification o f glutamine to the 2'-OH of the terminal adenosine. To test this, tRNA 6In molecules were prepared by chemical modification o f the 3'-termini. Purified

tRNA, tin was treated with sodium metaperiodate. followed by treatment with base to remove the 3'terminal adenosine (see legend to table I). Dephosphorylation with calf intestinal phosphatase then removed the 3'-terminal phosphate, resulting in tRNACl"--CC, lacking the terminal AMP. The 3'terminus was then repaired, by reaction with yeast tRNA nucleotidyl-transferase with ATP (as a control), 2'-dATP or 3'-dATP (cordycepin triphosphate) followed by gel purification o f the 3'-end-repaired tRNA Gin species. The modified tRNAs were then tested as substrates for aminoacylation with glutamine by GInRS [26] and the ability to catalyze the ATP/PP, exchange reaction, the first step of the aminoacylation reaction [27]. As table I shows tRNACla--CC3'dA can

Table I. Interaction of 3'-end-modified tRNA GIn species with GInRS. The preparation of the end-iepaired tRNA ~ln species followed earlier protocols [67, 68]. Aminoacylations were carded out as follows: the reaction mixture (50 la!) containing 30 mM HEPES/KOH (pH 7.0), 25 mM MgCI~, 4 mM ATP, 4 mM DTT, 0.05-5 laM tRNA ~ln, 10-500 IlM [14C]GIn (specific activity 150 cpm/mol) and 0.013 pmol GInRS. The enzyme was preincubated with tRNA at 37°C for 30 min. The reaction was started by addition of the amino acid and incubated at 37°C. After !, 2, 5, 15 rain aliquots (10 I.tl) were withdrawn and spotted onto 3 mM filter disks. The disks were washed three times for 15 min with 5% trichloroacetic acid and 5 min with ethanol and dried before the radioactivity was counted. For poor tRNA substrates the amount of enzyme (1.32 pmol per reaction) and tRNA (10-100 laM) were increased. The ATP/PPi-exchange reaction mixture (50 ~1) contained: 2 mM sodium [32p]pyrophosphate, 0.3 mM Gin, 8 ktM tRNA GIn, 2 mM ATP, 25 mM KCI, 15 mM MgCI 2, 4 mM DDT, 30 mM Pipes/KOH (pH 6.3). The reaction was started by addition ofO.i IaM GInRS and lllCUO~lcld . . . . . . . . . . a.t . . 9 l C. Aliquots of 10 lal were taken after 1, 5, 10, 30 min and spotted onto charcoal filter disks (Schleicher and Schuell) which were washed twice (15 min each) in 1.5% trichloroacetic acid containing 40 mM unlabeled sodium pyrophosphate and once (5 min) in water. After drying the radioactivity was counted [27].

End-repaired tRNA species

Aminoacylation reaction K~ k, (IlM) (s-~)

ATP/PPi exchange KM (12M)

tRNAGIn-CCA

0.5

2.6

15.0

tRNAGin-CC

nda

nd

nd

tRNAGtn-CC2'dA

nd

nd

nd

tRNAGtn-CC3'dA

0.6

1.6

8.0

alndicates that substrate properties were too poor to allow determination of kinetic parameters under the conditions used [26, 27].

15(M

S Englisch-Peterset al

be fldly c h a f e d (to 1568 pmol/A260) with kinetic parameters similar to those of reconstituted tRNAfi~--CCA, In vivo made tRNA GIn had comparable charging characteristics (data not shown). However, the extent of glutaminylation of tRNA GIn-CC2'dA is almost undetectable (below 21 pmol/A260) and tRNAG~m-CC could not be charged. Kinetic parameters could not be measured under the conditions used for these two tRNA species. The results from the ATP/PP. exchange reaction (table I) also ,how that while both tRNA°n--CCA and tRNAGIn--CC3'dA are good substrates, the tRNA Gl"--- CC2'dA and tRNA °~"-CC are very poor substrates for the ATP~P~ exchange reaction and activity could not be detected under these conditions. The lack of charging of tRNAr~--CC and tRNAG~n-CC2'dA was not caused by the inability of these RNAs to form a complex with GInRS; gel-shift experiments showed that all four tRNAs formed a stable complex (data not shown). The results from the end-repaired tRNA G~n species then show that GInRS has a strong preference for catalysis of aminoacylation of glutamine to the 2'-OH of the terminal adenosine. This correlates with the homology of GInRS to other members of the class I aminoacyl-tRNA synthetases [23]. However, in common with two other class I enzymes, ArgRS and GIuRS from E coli, GInRS cannot catalyze ATP-PP~ exchange in the absence of tRNA ([28]; summarized in [291). The absence of any detectable ATP/PP~ exchange reaction with tRNAGI"--CC and tRNA ClnCC2'dA is perhaps surprising, and indicates a requirement for both the terminal adenosine and the 2'-OH of the terminal adenosine of tRNA in the active site of t_he enz~.~n..e for A.TP~P, catalysis to occur. This ..'nay be because deformation of the 3'-end of tRNA Cln occurs upon binding to the enzyme, with the first base pair interrupted and a hairpin formed by the 3'terminal -GCCA to place the 3'-end in the active site of the enzyme [7]. The members of the class II synthetases show a strong preference for aminoacylation at the 3'-OH. The X-ray crystal structure of a class lI synthetase bound to tRNA, yeast AspRS: tRNAA~p, does not show the deformation of the 3' end of the tRNA, with the tRNA continuing a regular helical conformation when bound to the enzyme [30]. The deformation of the 3'-end of tRNA G~n when bound to the enzyme is then probably an example of induced fit [31] as E coli tRNA GIn and yeast tRNAA~p have high sequence homology in the acceptor stem region. It remains to be seen whether deformation of the tRNA is a common feature of the mechanism of aminoacylation by other members of class I synthetases; evidence from E coli MetRS [32], and E coli GIuRS for example indicates that this may be the case [33].

The identity elements of E coli tRNA tan

The set of nucleotides in the tRNA molecule that confer specificity by aminoacyl-tRNA synthetases are termed the identity eleraents (reviewed in [3, 34, 35]). Earlier genetic and biochemical studies have indicated that the identity elements of tRNA Gin are located in the anticodon [36-38] and in the acceptor stem [5, 39, 40] including the discriminator nucleotide [41]. The crystal structure of the tRNAGtn:GInRS complex defines in molecular detail the identity elements of tRNA GIn [7, 8]. To evaluate the contribution of the proposed identity elements, mutant tRNAs can be tested by in vivo suppression of an amber codon in the lacZ gene, lacZtooo [2, 5]. Mutations were made [42] of a synthetic gene for the amber suppressor supE derived from tRNA GIn and expressed in vivo [43]. A series of changes were made in the acceptor stem region of tRNA and the level of suppression determined by assay of ~-galactosidase activity, which is then proportional to the aminoacylation in vivo with glutamine. Mutations in the 3-70 base pair of supE have the largest effect on suppressor efficiency, and this is expected from specific recognition of G3 by GInRS [ 19]. The mutant supEG 1-C72 shows reduced suppression, probably from the strengthening of the first base pair that has to be disrupted in the complex with GinRS. Interestingly, mutations of the discriminator nucleotide (position 73) have little effect on suppressor efficiency, which is surprising from the requirement of G73 to maintain an intra-molecular hydrogen bond placing the 3' hairpin end of the tRNA in the active site of the enzyme. The advantage of the in vivo assay is that it is extremely sensitive. However~ mutations of the anticodon nucleotides cannot be studied, unless the tRNA is active in all steps of protein synthesis in vivo [5]. We then constructed another set of mutant tRNA o~n genes suitable for transcription in vitro and subsequent assay for aminoacylation by purified GInRS [44-46]. Mutation to a G at position l was necessary to ensure synthesis with T7 RNA polymerase, and this tRNAG~nG1 can be aminoacylated slightly better than tRNAG~"UI, made by cleavage of a larger precursor with the M1RNA component of RNase P (fig 2) [47]. As is the case for other tRNAs [45], the unmodified tRNA GIn is aminoacylated about one-third the level of in vivo tRNAG1% and the modified nucleotides enhance the reaction rate, perhaps by ensuring a tighter packing of the tRNA structure to fit more closely with the enzyme [8, 48]. This then allowed construction of a number of mutant tRNA ~n genes [42], which can be tested for aminoacylation with GlnRS [46] (fig 2). The results demonstrated that the largest effect of the mutations is on kcal values, with a smaller effect on K M. Correspondingly, the biggest

tRNAGIninteraction

UI 4,-- G

cGAA

A C C G ~ A ~

C/U72 */~

* A 2 ---- G - C ~ U71*** w,~*A3 ----- G - C ---,-- U 7 0 ~ * * G-C G-C U-A A-!J C U GCUCC UA CCG

G

AICIU73~I~I.~.~

,,,

, , ,

,,

~....,rc-Ar-c- U

GU A A G G C A

CU

U

A G C

C-GCAu C G G-C G-C A - U ...~G:L58 * * U U U A ~U37 *~ C G A 3 4/

u,, A C35

i 505

The modified nucleotides m-~A37 and W38 may then stabilize the conformation of the anticodon nucleotides, as has been suggested for other tRNAs (reviewed in [49]). Mutations in the acceptor stem of tRNAGInGI have a smaller effect on the kinetic parameters for aminoacylation by GInRS. Alteration of the first base pair leaves aminoacylation by GInRS virtually unaffected, while strengthening the base pair in tRNA~I"GIC72 reduces catalytic efficiency with GInRS 10-fold (fig 2). This supports the results from mutations made in the first base pair in tRNA rM~t and the effect on aminoacylation by GlnRS [50], as well as the results from the amber suppressor supE. However, apart from A73, mutations at the discriminator nucleotide to C or U reduce k~JK M 1700- and 300-fold respectively. This contrasts with the small effect of these mutations on supE in vivo, and may be due to less conformational stability of the in vitro tRNA transcript. The largest effect of mutations in the acceptor stem of tRNAG~"G 1 is at base pairs 2-71 and 3-70, with G2 and G3 recognized specifically by GInRS [19]. Therefore, the sequence and precise location of the G2--C71 and G3-C70 base pairs is important for specific recognition by GInRS.

Fig 2. The sequence of tRNA GIn shown in cloverleaf form,

with the numbered nucleotides indicating mutations made in supE for in vivo expression of tRNAGIn and in tRNAGInG1 for in vitro transcription. The approximate value of the relative specificity constant (K~,/KM) determined by aminoacylation [46] is indicated by *. The scale decreases one log with each * (eg * is 10-1, **** is 10-~) and is set to I for tRNAGInGI. impairment to catalysis is from mutations in the anticodon (positions 34-36). The most severe reduction is from the pyrimidine (C35) substitution at the central position of the anticodon, a position implicated by previous genetic studies and confirmed by sequencespecific recognition of U35 in the crystal structure [8]. Surprisingly, tRNACI,G IA36, which now has the anticodon corresponding to an amber suppressor tRNA, is also an extremely poor"substrate for aminoacylation in vitro, although this is the functional glutamine amber suppressor supE in vivo. The in vivo suppression assay is then very sensitive, requiring a small threshold value of aminoacylation for suppression. Since each anticodon nucleotide is specifically recognized by GInRS, the mutations at these positions in tRNAGI"GI confirm the discrimination by GInRS of the anticodon. Additionally, mutations at positions 37 and 38 of the anticodon loop also reduce aminoacylation by GInRS, possibly by affecting base-pairs 33-37 and 32-38 in the extended anticodon stem observed in the complex of tRNA ~l~ with GInRS [8].

Shared_ p o s i t i o n s o f i d e n t i t y e l e m e n t s in t R N A ?

Recent progress in determining the structural features of tRNA recognition has been made for various tRNAs by in vitro aminoacylation and by in vivo suppression: experiments in E coil using the Alasystem [51, 52]; the Gin-system [5]; the Phe-system t J I ; t . e o~t-~yatel, i-r_,, .-,a|, t,,,.. ,,.,..~ o ro,~.,, t~ ,j, the yeast Asp-system [55-57]; the Arg-system [58, 59]; and in yeast the Phe system [45]. In general, the acceptor stem nucleotides and the anticodon have been shown to be of importance to tRNA identity. Additionally, in tRNA eh~ and tRNAAr~ another supporting element to tRNA identity was found to be with the 'variable pocket" of the D-loop/T-loop-interaction, and in tRNA s~r the variable loop (extra arm) is important. Thus four regions in the tRNA (anticodon, acceptor stem, variable pocket and extra arm) are implicated to date in cognate synthetase:tRNA interaction. It is interesting to note that already over two decades ago some of these regions were implicated as important for tRNA discrimination by synthetases. Early experiments from the inhibition of aminoacylation by synthetic oligoribonucleotides of defined sequence indicated the importance of specific recognition of the anticodon of tRNA by aminoacyl4RNA synthetases [60]. Reconstitution of aminoacylation activity (albeit low) from fragments of yeast tRNA A~a

150(~

S Englisch-Peters et a/

1611 showed the importance o f the acceptor stem for recognition in this system: experiments recently and elegantly extended into new areas by m o d e m techniques 1621.

2

3 4

Outlook The availability of mutant tRNA and GInRS molecules, together with the structural analysis o f the mutants will define in molecular detail the discrimination by GinRS. Currently, we have identified bases in the anticodon and acceptor stem regions of t R N A C~" important for specificity. Other positions may include the positions G I 0 and C16 of tRNA ~n" [7]. Further experiments to transfer these identity elements to different tRNA backgrounds should reveal the importance and additive effect o f each element. However, other bases important for tRNA Cnn identity m a y be positions not in direct contact with the e n z y m e but which help to keep the correct tRNA structure, and which also block recognition by other aminoacyltRNA synthetases: large modified nucleotides may play an important negative role in GinRS recognition [81, as is the case for a modification in the anticodon o f t R N A ne that prevents recogn~,tion by MetRS [63]. The specific recognition by GInRS may involve a long-range transmission o f specific recognition o f the anticodon to the active site o f GInRS. Genetic selection for mutants o f GInRS may reveal the nature o f this effect, which has been successful for genetic selection for anticodon binding mutants of E coil MetRS [64], acceptor stem binding mutants o f E coil AIaRS [65] and mischarging mutants o f GInRS (summarized in [3]). It is evident that efficient aminoacylation o f t R N A is a compromise between Iunction in protein synthesis at a rapid rate and at a high level o f accuracy. This suggests that in fact the t R N A is probably 'overdiscriminated" by the synthetase to ensure accuracy o f aminoacylation, and competition in vivo between the synthetases is a major factor in this [4, 66].

Acknowledgments

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,L.,OL ttut

14 15

16

We would like to thank all the members of our group for discussions and support. This work was funded by a grant from NIH. 17

References Schimmel P (I 987) Aminoacyl tRNA synthetases: general scheme of structure-function relationships in the polypeptides and recognition of tRNAs. Annu Rev Biochem 56. 125-158

Inokuchi H, Hoben E Yamao F, Ozeki H, Still D (1984) Transfer RNA mischarging mediated by a mutant Escherichia coli glutaminyl-tRNA synthetase. Proc Natl Acad Sci USA 81,5076-5080 Rogers MJ, SOll D (1990) Inaccuracy and the recognition of tRNA. Prog Nucleic Acid Res Mol Bio139, 185-208 Swanson R, Hoben P, Sumner-Smith M, Uemura H, Watson L, SOIl D (1988) Accuracy of in vivo aminoacylation requires the proper balance of tRNA and aminoacyltRNA synthetase. Science 242, 1548-1551 Rogers MJ, Still D (1988) Discrimination between glutaminyl-tRNA synthetase and seryl-tRNA synthetase involves nucleotides in the acceptor helix of tRNA. Proc Natl Acad Sci USA 85, 6627--6631 Perona JJ, Swanson R, Steitz TA, Still D (1988) Overproduction and purification of Escherichia coli tRNA,Ctn and its use in crystallization of the glutaminyl-tRNA synthetase:tRNAGIn complex. J Mol Bio1202, 12 I-I 26 Rould MA, Perona JJ, Still D, Steitz T (1989) Structure of E coli glutaminyl-tRNA s2¢nthetase complexed with tRNAtin and ATP at 2.8 A resolution. Science 246, 1135-1142 Rould MA, Perona JJ, Steitz TA (I 991) Structural basis of anticodon loop recognition by glutaminyl-tRNA synthetase. Nature 352, 213-218 KOrner A, Magee BB, Liska B, Low KB, Adelberg EA, $611 D (1974) Isolation and partial characterization of a temperature-sensitive Escherichia coli mutant with altered glutaminyl-transfer ribonucleic acid synthetase. J Bacteriol 120, 154-158 Yamao F, Inokuchi H, Cheung A, Ozeki H, Still D (1982) Escherichia coil glutaminyl-tRNA synthetase, I. Isolation and DNA sequence of the ginS gene. J Biol Chem 257, 11639-11643 Isaksson LA, Skold SE, Skjoldebrand J, Takata R (1977) A procedure ~for i~olaticn of spontaneous mutants with temperature-sensitive synthesis for RNA and/or protein. Mol Gen Genet 156, 233-237 Cheung AY, Still D (1984) In vivo and in vitro transcription of the Escherichia coli glutaminyl-tRNA synthetase gene. J Biol Chem 259, 9953-9958 Uemura H, Conley J, Yamao F, Rogers J, SOll D (1988)

18

lq. tttt,t

LOll

~IUI.dililII~/l--tl[N.l~l,t

~-

amino acid replacement relaxes tRNA specificity. Prowin Sequences & Data Anal I, 479-485 Conley JG (1988) Mutational analysis of Escherichia coli glutaminyl-tRNA synthetase. PhD Thesis, Yale University Morgan S, Ktirner A, Low KB, Still D (1977) Regulation of biosynthesis of aminoacyl-tRNA synthetase and of tRNA in Escherichia coll. I. Isolation and characterization of a mutant with elevated levels of tRNA, Gl~. J Mol Biol 117, 1013-1031 Cheung A, Morgan S, Low KB, Still D (1979) Regulation of the biosynthesis of aminoacyl-transfer ribonucleic acid synthetases and of transfer ribonucleic acid in Escherichia coli. VI. Mutants with increased levels of glutaminyl-transfer ribonucleic acid synthetase and of glutamine transfer ribonucleic acid. J Bacteriol 139, 176-184 Caillet J, Plumbridge JA, Springer M, Vacher J, Delamarche C, Buckingham RH, Grunberg-Manago M (1983) Identification of clones carrying an E coil tRNA ene gene by suppression of phenylalanyl-tRNA synthetase thermosensitive mutants. Nucleic Acids Res I 1,727-736 Perona JJ (1990) Crystal structure of the E coli glutaminyl-tRNA synthetase:tRNA GIncomplex. PhD Thesis. Yale University

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3! 32

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