tRNA residues that have coevolved with their anticodon to ensure uniform and accurate codon recognition

Biochimie 88 (2006) 943–950 www.elsevier.com/locate/biochi

tRNA residues that have coevolved with their anticodon to ensure uniform and accurate codon recognition Mikołaj Olejniczak1, Olke C. Uhlenbeck* Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA Received 26 January 2006; accepted 2 June 2006 Available online 23 June 2006

Abstract The structure, phylogeny and in vivo function of the base pair formed between nucleotides 32 and 38 of the tRNA anticodon loop are reviewed. The A32–U38 pair, which is highly conserved in tRNA2Ala and sometimes observed in tRNA2Pro, was recently found to decrease the affinity of tRNAs to the ribosomal A site relative to other 32–38 combinations. This suggests that the role of 32–38 pair is to tune the tRNA affinity in the A site to a uniform value. New experiments presented here show that the U32C mutation in tRNA1Gly increases its affinity to the cognate codon and to codons with third position mismatches in the A site. This suggests that one reason for uniform tRNA binding to evolve was to avoid incorrect codon recognition. © 2006 Elsevier SAS. All rights reserved. Keywords: Translation; Decoding; Two-out-of-three reading; tRNA uniformity; tRNA tuning

1. Introduction

2. Structures of the 32–38 pair

Residues 32 and 38 in tRNA form a non-canonical base pair at the top of the anticodon loop, adjacent to the anticodon stem [1] (Fig. 1). Because of its critical position near the anticodon, this base pair has been implicated in many aspects of tRNA function including intron splicing [2], several posttranscriptional modifications [3] and aminoacylation [4]. The focus of this paper is to consider the role of the 32–38 pair in the process of decoding on bacterial ribosomes. After briefly reviewing relevant structural, phylogenetic, and functional data, the consequences of mutations in the 32–38 pair upon binding of two different Escherichia coli tRNAs to the A site will be considered in detail. We propose that an important role of this pair is to adjust how tightly aminoacyl-tRNAs (aatRNAs) bind to the ribosome. This adjustment is needed to ensure that all aa-tRNAs bind ribosomes with a similar affinity, despite large inherent differences in the strength of the anticodon–codon interaction.

A comparison of more than 5600 bacterial tRNA gene sequences [5] reveals an uneven distribution of 32–38 pairs with C–A, U–A and U–U being the most common and pairs containing G being the least common (Table 1). As reviewed by Auffinger and Westhof [1], much of this distribution can be understood by the structures assumed by the 32–38 pairs. Based on 28 crystal structures of three free tRNAs and 10 tRNA-protein complexes, the majority of 32–38 pairs have been assigned to two different families of non-Watson–Crick base pairs. Pairs in each family are isosteric to one another but not to the representatives of the other family. Family I includes C–A, U–C, U–A and C–C pairs as well as versions containing modified residues. Examples of the Family I include the Cm32–A38 [6] (Fig. 1A), which forms a bifurcated hydrogen bond between the O2 of cytidine and the N6 of adenosine, and the isosteric Ψ32–C38 pair (Fig. 1B), where a similar bifurcated hydrogen bond is formed between the O4 of pseudouridine and the N4 of cytidine [7]. Family II exclusively contains the U32–U38 pair (Fig. 1C) and the related pairs containing pseudouridines. In this case a single hydrogen bond forms between the N3 of uridine 32 and the O4 of uridine 38 [8]. Because more than 90% of all 32–38 pairs belong to one of

* Corresponding

author. E-mail address: [email protected] (O.C. Uhlenbeck). 1 Present address: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland. 0300-9084/$ - see front matter © 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2006.06.005

944

M. Olejniczak, O.C. Uhlenbeck / Biochimie 88 (2006) 943–950

Fig. 1. Structures of 32–38 pairs. a) Cm32–A38 from the crystal structure of free yeast tRNAPhe (pdb entry: 1EHZ) [6], b) Ψ32–C38 from the crystal structure of free yeast tRNAAsp (pdb entry: 3TRA) [7], c) U32–U38 from the crystal structure of the complex of tRNAGln with glutaminyl-tRNA synthetase (pdb entry 1O0C) [8], d) U32–A38 from the crystal structure of E. coli ASLPhe in the A site of T. thermophilus 30S subunit (pdb entry 1IBM) [10], e) Ψ32–A38 from the crystal structure of the complex of E. coli tRNACys with T. aquaticus EF-Tu (pdb entry 1B23) [11], f) U32–A38 from the crystal structure of the complex of tRNAPro (CGG) with prolyl-tRNA synthetase (pdb entry 1H4Q) [12]. Table 1 32–38 base pairs in tRNAa 32–38 pair CA UA UU CC UC AU AA UG CG AC CU GA GU AG GG GC

Total number 2928 962 608 470 261 113 83 75 67 12 9 8 3 1 1 0

% 52.3 17.2 10.9 8.4 4.7 2.0 1.5 1.3 1.2 0.21 0.16 0.14 0.05 0.02 0.02 0.00

Family I I II I I III III III III III III III

Structure + + + – + – – – – – – – – – – –

a

Base-pair occurrence in all 5601 bacterial tRNA genes available in a tDNA database [5].

these two families, it was proposed that the structure of the pair is essential for anticodon loop to assume its canonical, U-turn conformation [1,9]. Interestingly, the U32–A38 pair, which has been included in Family I, can assume conformations that are not isosteric with other family members. The U–A pair present in the unmodified anticodon stem-loop (ASL) of E. coli tRNAPhe bound in the A

site of the Thermus thermophilus 30S subunit could form a bifurcated hydrogen bond typical of Family I [10] (Fig. 1D). However, in the X-ray structure of tRNACys complexed with elongation factor Tu, the N6 of A38 faces the C6 instead of the O4 of Ψ32 and thus the hydrogen bond cannot form [11] (Fig. 1E). Moreover, the U32–A38 pair forms a classic, Watson–Crick conformation in the crystal structure of tRNAPro complexed with its synthetase [12] (Fig. 1F). Finally, in contrast to when it is bound to the ribosome, the structure of the unmodified ASLPhe free in solution determined by NMR [13] also possesses a conventional U32–A38, Watson–Crick pair. Thus, the structure of a 32–38 pair may change when a tRNA binds to proteins or to different sites on the ribosome. The approximately 6% of 32–38 pairs that are not in either Family I or II were grouped as a Family III [1]. It is likely that these do not form a homogenous group, but a number of different structures. However, it is striking that even though they are not isosteric to the mainstream pairs, several of these rare outliers are conserved and specific for a given tRNA isoacceptor. As mentioned above, the crystal structure of the ASLPhe bound to 30S ribosomal subunit reveals that the conformation of U32–A38 pair has the typical Family I structure [10] (Fig. 1D). Although extensive contacts between anticodon– codon helix and the 16S rRNA residues A1492, A1493 and G530 are observed, residues 32 and 38 do not directly contact

M. Olejniczak, O.C. Uhlenbeck / Biochimie 88 (2006) 943–950

945

Table 2 Examples of 32–38 pair correlation with anticodon sequencea tRNA (anticodon)

32–38 pair (frequency)

tRNA1Ala (UGC)

U–C (37%), U–A (33%), C–C (19%), U–U (6%) A–U (77%), C–G (22%) C–C (69%), U–U (14%), C–A (11%) U–A (98%) C–A (98%) U–A (97%) U–U (50%), U–G (47%) C–A (99%) C–A (62%), U–A (24%), U–C (12%) C–G (48%), A–U (34%), U–U (15%) U–A (60%), A–A (38%) U–U (63%), C–C (14%), U–G (8%), U–C (8%)

tRNA2Ala (GGC) tRNAGlu (UUC) tRNA1Gly (CCC) tRNA2Gly (UCC) tRNA3Gly (GCC) tRNA2Leu (GAG) tRNALys (UUU) tRNAPhe (GAA) tRNA2Pro (GGG) tRNAThr (GGU) tRNA2aVal (GAC)

Fig. 2. The environment of ASLPhe in the A site of T. thermophilus ribosomal 30S subunits (pdb entry 1IBM) [10]. ASL residues are presented in red, mRNA residues in light blue and 16S rRNA residues in dark blue.

any rRNA residues in the A site (Fig. 2). On the other hand, even though the structure of yeast tRNAPhe bound to the P site is not available at as high resolution, it appears that the Cm32– A38 pair also has a Family I conformation, and that the ASL forms several contacts with 16S rRNA [14]. In the higher resolution 30S structure, the stem-loop of helix 6 from another ribosome in the crystal lattice appears to mimic the ASL of P site tRNA and also forms contacts with the 16S rRNA [15]. The conserved 16S rRNA nucleotide A790 interacts with the backbone of tRNA residue 38 and perhaps 37. Additional hydrogen bonds are formed between the conserved 16S rRNA nucleotides G1338 and A1339 and the nearby tRNA anticodon-stem nucleotides 40 and 41. While substitutions of G1338 and A1339 are highly detrimental for in vivo translation, substitutions of A790 only moderately decreased translation activity suggesting that its binding to tRNA is not crucial for tRNA function on the ribosome [16]. In any case, the ASL is clearly recognized quite differently by the ribosome in the A and P sites, and consequently the role of 32–38 pair in these sites is expected to be different. 3. Anticodon-specific phylogeny of the 32–38 pair The observation that a tRNA anticodon correlated with sequences in the ASL was first pointed out by Yarus [17]. On the basis of a limited number of tRNA sequences, his “extended anticodon” hypothesis proposed that residues 37 and 38 on the 3′ side of the anticodon, as well as several pairs in the anticodon stem, had evolved predominantly to suit the identity of the nucleotide in the position 36 of the anticodon which he termed the cardinal nucleotide. Mutagenesis experiments with suppressor tRNAs showed that this correlation serves to maximize the translational efficiency of the tRNA [18].

Number of sequences 116 73 104 50 103 101 92 103 105 67 97 79

a For the analysis, only non-redundant bacterial tRNA gene sequences from a tDNA database [5] were included. Only those pairs that occur in at least 5% and five sequences of a particular tRNA isoacceptor are presented. Pairs shown in bold, italicized font are rare, and occur in 2% or less of all bacterial tRNA gene sequences.

The availability of a large number of bacterial genome sequences [19] now makes it possible to further elaborate and somewhat modify the concept of the extended anticodon. An analysis of sequences of each individual tRNA isoacceptor reveals a phylogenetic correlation between all three anticodon residues and a number of residues throughout the entire tRNA body that are different for each isoacceptor (M.E. Saks, submitted). Thus, not only do tRNAPhe and tRNA1Gly differ, but also each of the three tRNAGly isoacceptors have significantly different consensus sequences. In other words, each tRNA body appears to have evolved to match its anticodon. This pattern of isoacceptor-specific sequence conservation often includes a very strong correlation between the anticodon sequence and the identity of one or few 32–38 pairs [20]. As summarized in Table 2, some isoacceptors always have the same 32–38 pair, such as U–A in tRNA1Gly or C–A in tRNALys, while others have a specific subset of combinations such as the rare A–U and C–G pairs present in tRNA2Ala and tRNA2Pro. Finally, for some isoacceptors such as tRNA1Ala and tRNA2aVal, the 32–38 positions vary considerably. However, even in these cases, the pattern of the conserved 32–38 pairs is remarkably different than the average (Table 1) and specific for a given anticodon. These phylogenetic results suggest that the identity of the 32–38 pair in some way correlates with the identity of the anticodon trinucleotide. 4. Function of the 32–38 pair in vivo There is abundant evidence from the analysis of suppressor tRNAs that the identity of the 32–38 pair affects tRNA function in vivo. After initially finding that tRNA suppressor efficiency is primarily defined by the ASL [21,22], many mutagenesis experiments revealed that positions 32 and 38 modulate the translational performance of UAG, UAA and UGA suppressor tRNAs [18,22–26]. As summarized in Table 3 for six different UAG (amber) suppressor tRNAs, changing the

946

M. Olejniczak, O.C. Uhlenbeck / Biochimie 88 (2006) 943–950

Table 3 The effect of 32–38 pair changes on suppressor efficiencies tRNA tRNA1Ala tRNA2Ala tRNAGlu tRNA2Gln tRNAHis tRNATrp tRNATrp a

Relative effect of 32–38 change on suppression efficiencya U–U < U–C < C–C < C–A A–U < A–A C–C < C–A Um-Ψ < Um–C < Um–A < Cm–A U–U < U–A Um-Ψ < Um–C < Um–A < Cm–A C–G < G-A < U-G < A–A < CU < U–A < C–C < C–A

References [25] [23] [24] [22] [24] [22] [18]

The 32–38 residues in each parental tRNA are italicized.

32–38 pair present in the parental tRNA was found to either improve or diminish suppressor efficiency. Interestingly, while suppressor tRNAs differed substantially in the overall efficiency of suppressing an UAG mutation, they all showed similar order with respect to the 32–38 pair, namely UU < UC < UA < CC < CA. In other words, the suppressor tRNAs worked best with C32–A38 and worst with U32–A38 irrespective of the pair present in the parental tRNA. However, since these experiments were done with the amber anticodon, it is unknown whether this hierarchy of 32–38 effects would be true for other anticodons. Data evaluating the effect of changing positions 32 and 38 on the performance of natural, non-suppressor tRNAs in vivo are very limited. It is known that over-expressing the U32C mutation in tRNA1Gly in cells containing a normal copy of the tRNA1Gly gene results in the increased efficiency of frameshifting [27]. A recent series of experiments using a tRNA gene replacement strategy showed that nucleotide changes at 32 and 38 in tRNAThr influence the growth of strains which contain the mutant tRNA (W. Liu, M.E. Saks, submitted). These data, although scarce, suggest that the identity of the evolved 32–38 pair is also important for the in vivo translational function of natural, elongator tRNAs, but no specific rules have emerged. 5. The 32–38 pair ensures uniform tRNA binding to the A site It was recently observed that aminoacylated, fully modified tRNAs have very similar affinities to both the A and P sites [28]. However, when either the esterified amino acid or the nucleotide modifications were removed, the binding was no longer uniform. It was proposed that tRNA sequences and their post-transcriptional modifications had evolved to compensate for the differing thermodynamic and kinetic contributions associated with the amino acid and anticodon. As a result each aatRNA could be used equivalently by the translational apparatus [29,30]. Clear evidence that the 32–38 pair participates in adjusting tRNA affinity to the ribosomal A site emerged from an analysis of foreign anticodons transplanted into tRNA2Ala [20]. Purified Ala-tRNA2Ala binds to its cognate GCC codon in ribosomal A site with similar affinity as previously found for eight other aa-tRNAs binding to their cognate codons [28,31]. However, when seven anticodons were transplanted into tRNA2Ala, all resulting chimeric tRNAs bound their comple-

mentary codons in the A site much less well than either tRNA2Ala or any of the wild type tRNAs containing the same anticodons [20]. Subsequent sequence dissection experiments revealed that tight ribosomal binding of tRNA2Ala (GCC) and tRNA2Ala (ACG) was restored when A32–U38 was replaced by either the U32–A38 or C32–A38 combinations normally associated with the GCC and ACG anticodons, respectively (Fig. 3). Thus, the A32–U38 pair weakens the binding of the chimeric tRNAs to the ribosome. Further experiments revealed that the rare A32–U38 pair [32] also weakens the binding of the natural tRNA2Ala (GGC) to its cognate GCC codon [20]. When this pair was replaced by U–A, U–U and A–A combinations, the binding became substantially tighter than for the wild type tRNA2Ala. However, when the pair was replaced by phylogenetically observed alternate C32–G38 pair, which is present in the tRNA2Ala sequences of about 25% of bacterial species, the binding was unchanged. We speculated that because of the exceptional stability of the GGC–GCC anticodon–codon pair tRNA2Ala has evolved to have 32–38 pairs that weaken its ribosomal binding in order for its value to be similar to other tRNAs. Interestingly, bacterial tRNA2Pro sequences, which have another stable GGG-CCC anticodon–codon pair, also have either the A32– U38 or the C32–G38 pair to destabilize tRNA binding (Table 2). Thus, the identity of the 32–38 pair may also be used by other tRNAs to tune the anticodon–codon affinities on the ribosome. 6. The 32–38 pair and third position misreading Another well studied example of the effect of modifying the 32–38 interaction on tRNA function was showed in a series of in vitro translation experiments using E. coli and Mycoplasma mycoides glycine tRNAs [33–35]. In E. coli there are three tRNAGly isoacceptors: tRNA1Gly (CCC) which recognizes exclusively the GGG codon, tRNA2Gly (UCC) which reads the GGA and GGG codons and tRNA3Gly (GCC) which reads the GGC and GGU codons. On the other hand, M. mycoides has only one glycine tRNA with UCC anticodon, which therefore must be capable of reading all four glycine codons. Indeed, in vitro translation experiments showed that the three E. coli glycine tRNAs did discriminate among glycine codons as expected, while M. mycoides tRNAGly was almost as efficient in misreading GGU and GGC codons as it was in the conventional GGA and GGG codons reading [36]. These experiments evaluated the relative reading efficiencies of these tRNAs in competition assays in which one tRNA was esterified with 3H glycine and the other with 14C glycine. The relative efficiency of two tRNAs in reading of a given glycine codon could then be established based on the isotopic ratio of glycine residues incorporated into peptide. Using this system, it was established that discrimination of the third codon position depended on the identity of nucleotide 32. When the U32 of E. coli tRNA1Gly was changed to a C, it lost the ability to discriminate among glycine codons [34,35]. Similarly, when the C32 present in wild-type M. mycoides tRNAGly (UCC) was changed to U, it only was capable of efficiently reading the

M. Olejniczak, O.C. Uhlenbeck / Biochimie 88 (2006) 943–950

947

Fig. 3. Relative KD values for derivatives of unmodified E. coli tRNA2Ala calculated from Ref. [20]. A value of 1.0 corresponds to KD = 160 nM and is similar to many different E. coli aa-tRNAs. The A32–U38 pair and the C32–G38 pair present in some species weaken tRNA binding relative to other combinations.

GGA codon [35]. Thus, in two different tRNA backgrounds, the ability to discriminate among glycine codons depended on the presence of U32. When C32 was present, the discrimination was lost. The above competition studies measure the overall efficiency of translation, but do not explain how the third position misreading is affected by the identity of nucleotide 32. Because our previous studies [20] had shown that mutations of 32–38 pair can affect the tRNA affinity to the ribosome, we decided to test E. coli tRNA1Gly and tRNA1Gly (U32C) binding to cognate codons and third position mismatches in the ribosomal A site. Similar to tRNA2Ala, tRNA1Gly does not contain modifications in the ASL and glycine is also a small amino acid, suggesting that the modifications and the presence of an esterified amino acid would not be important for binding of this tRNA to the A site. Indeed, unmodified, deacylated tRNA1Gly was found to dissociate from its GGG codon with a koff value of 11.7 × 10−3 min−1, which is similar to the koff values of most fully modified aminoacyl-tRNAs [28]. This permits the use of unmodified, deacylated tRNAGly derivatives to explore the effect of position 32 on third position misreading. The affinity of wild type tRNA1Gly and mutant tRNA1Gly U32C to the cognate and near-cognate codons in the A site of

E. coli ribosomes was measured using the dissociation rate and equilibrium binding assays performed in a dual filter, 96-well plate format [37]. The dissociation rate experiments (Fig. 4A), show that the rate of dissociation of tRNA1Gly is reduced only twofold when U32 is changed to C32. However, markedly larger differences are observed between the two tRNAs when dissociation rates from near-cognate codons are examined. While filter stable complexes between tRNA1Gly and the near-cognate GGC, GGA and GGU codons could form, the dissociation rate was too fast to measure using a manual pipetting assay, and hence must dissociate from these mismatched codons at least 50-fold faster than from the cognate GGG codon. In contrast, the mutated tRNA1Gly (U32C) dissociated from the three mismatched codons much more slowly with a rate only about 12fold faster than dissociating from its cognate GGG codon. As summarized in Table 4, the dissociation rate data clearly show that a C32 in tRNA1Gly selectively stabilizes tRNA binding to codons with mismatches in the third position. In order to confirm the dissociation rate data, an equilibrium binding assay was also used to measure the affinity of wildtype and U32C mutant tRNA1Gly to the four codons. Previous experiments assaying many different aa-tRNAs to either the ribosomal A and P site have shown that the ratio of the disso-

Table 4 Dissociation rates of tRNA1Gly and tRNA1Gly (U32C) from E. coli ribosomes A site P site

Codons: tRNA1Gly tRNA1Gly (U32C) tRNA1Gly tRNA1Gly (U32C)

GGG 11.7 ± 1.8 6.1 ± 1.7 6.8 ± 1.9 4.7 ± 0.5

All koff values are times 103 × min−1. Buffer conditions are given in Fig. 4.

GGC >500 65 ± 20 6.9 ± 0.5 5.1 ± 0.7

GGA >500 81 ± 29 6.3 ± 1.2 4.5 ± 0.4

GGU >500 51 ± 14 6.8 ± 1.6 4.0 ± 1.2

948

M. Olejniczak, O.C. Uhlenbeck / Biochimie 88 (2006) 943–950

an in vitro translation assay [35] is that the mutation in some way stabilized tRNA binding to ribosomes. This effect is most evident when a third position mismatch is present. Interestingly, neither tRNA1Gly nor the U32C mutants can distinguish between cognate and near-cognate codons in the P site (Table 4). Although tRNA1Gly (U32C) binds to a P site codon slightly tighter than tRNA1Gly, the affinity of wild-type and mutant tRNA to cognate and near-cognate codons is the same. This observation underscores differences in tRNA binding to the A and P site. 7. Possible mechanisms of tuning by the 32–38 pair

Fig. 4. The U32C mutation in tRNA1Gly results in tighter binding to both cognate GGG and near-cognate GGC, GGA and GGU codons in the A site of E. coli 70S ribosomes. a) A site dissociation rates of wild type tRNA1Gly (black) and tRNA1Gly U32C (red) from GGG (squares), GGC (diamonds), GGA (triangles) and GGU codons (inverted triangles). Average koff values are given in Table 4. Dissociation kinetics were determined as previously described in [37] in ribosome binding (RB) buffer (50 mM Hepes pH 7.0, 70 mM NH4Cl, 30 mM KCl, 10 mM MgCl2 and 1 mM DTT) at 20 °C. For all experiments the complex was formed at 1000 nM ribosomes and dissociation initiated by diluting to 8 nM ribosomes. b) Equilibrium A site binding of tRNA1Gly (black) and tRNA1Gly U32C (red) binding to GGG (squares), GGC (diamonds), GGA (triangles) and GGU codons (inverted triangles). Lines are best-fit KD values of 160 nM for tRNA1Gly on GGG codon and 21, 190, 290 and 210 nM for tRNA1Gly U32C on GGG, GGC, GGA and GGU codons, respectively. KD values for tRNA1Gly on GGC, GGA and GGU codons were all above 1 μM. Equilibrium binding was performed as previously described in [20], with samples equilibrated for 4 hours in RB buffer at 20 °C prior to filtration. The data were fit to a hyperbolic equation using GraphPad Prism software (GraphPad Software).

ciation rate constant and equilibrium binding remains constant, indicating a constant association rate constant [37,38]. This makes it possible to use the equilibrium binding assay in cases where the dissociation rate is too fast to measure accurately. As shown in Fig. 4B, the effect of the U32C mutation slightly stabilized the binding to the three mismatched codons. Thus, the trends observed with the koff assay are confirmed by the equilibrium binding assay. Taken together the two assays indicate that tRNA1Gly U32C has a higher affinity to nearcognate codons than wild type tRNA1Gly. Therefore, a reasonable explanation why tRNA1Gly U32C is unable to efficiently discriminate cognate codons from third position mismatches in

When the U32–A38 pair is changed to C32–A38 in tRNA1Gly or when A32–U38 pair is changed to U32–A38 in tRNA2Ala, the affinities of these tRNAs to the ribosome increase. While it was initially suggested that nucleotides 32 and 38 could modulate translational performance of tRNA by directly interacting with ribosomal residues [21,39], the high resolution crystal structure of the A site does not show any direct contacts between 32 or 38 nucleotides and the ribosome [10]. Indeed, biochemical experiments showed that in the A site tRNA strongly protects only 16S rRNA nucleotides G529, G530, A1492 and A1493 [40], which are all located in the vicinity of the anticodon–codon duplex and far from 32–38 pair [10]. Thus, the available structural and biochemical data do not indicate any direct interactions between the 32–38 base pair and 16S rRNA in the A site. It is possible that the identity of the 32–38 pair could exert its effect on tRNA affinity to the ribosome indirectly, by modulating the anticodon loop conformation in the free form. Indeed, the solution NMR studies of unmodified ASLPhe and ASLLys, which have a decreased affinity to the ribosome, show that these ASLs form triloop hairpin structures where 32–38 and 33-37 nucleotides are Watson–Crick paired [13,41]. Dao et al. [42] have argued that in order to bind the ribosome, the tRNA anticodon loop needs to be in an “open” conformation where U33-A37 and U32–A38 nucleotides are not paired. Thus, any tRNA that is in the incompatible form in solution must pay an energetic penalty to bind. This could explain many of the 32–38 effects. For example, the energetic cost of breaking U32–A38 pair in order to achieve the “open” conformation is expected to be larger than in case of mismatched C32–A38 pair. As a result, tRNAs with the latter pair will bind tighter. If the effects of 32–38 modifications on ribosome binding were entirely due to the different propensity of free tRNAs to adopt an “open” conformation one would expect that their effect on the equilibrium constant would be on the association rate constant. Thus, a tRNA in the “closed” conformation would bind less well because it bound more slowly. However, the ribosome binding data presented here and elsewhere [20] show that the 32–38 mutations primarily affect ribosome binding by changing the dissociation rate constant. In cases where both KD and koff were measured, the ratio koff/KD = kon remained fairly constant. Thus, it appears that even less com-

M. Olejniczak, O.C. Uhlenbeck / Biochimie 88 (2006) 943–950

patible 32–38 combinations are able to bind ribosomes at a normal rate, but the stability of the complex is weaker, leading to more rapid dissociation. Given that the ribosome does not contact the 32–38 pair directly, how could this pair affect the dissociation rate in the bound form? One likely possibility is that these residues affect the details of the geometry of the nearby anticodon–codon helix. The minor groove of this helix forms extensive contacts with residues in 16S rRNA which are thought to be coupled to a large scale conformational change on the ribosome [10,43]. When mismatches occur in the anticodon–codon helix, the conformational change does not occur as early, permitting accurate decoding. It is also known that even small functional group changes in the anticodon loop can modulate ribosome dissociation rates [38,44]. Thus, residues 32–38 could modify the ease with which the ribosome undergoes this critical conformational change in the decoding process. 8. Conclusions The new experimental data presented here support the view that tRNAs evolved to bind the ribosome with uniform affinity in order to avoid incorrect codon recognition. If tRNAs bound ribosomes too tightly, mismatched codons would be recognized. It appears that the identities of 32–38 pairs coevolved with particular tRNA isoacceptors to compensate for different contributions of the anticodon sequence, esterified amino acid and modifications to the uniform ribosomal affinity. Even though the majority of 32–38 pairs found in tRNAs can be included into two isosteric families, the pairs belonging to a given group can assume different conformations depending on the interactions that they participate in. Moreover, several rare, but highly conserved 32–38 pairs can presumably assume conformations different from those typical for common pairs. This structural diversity allows the identity of 32–38 pairs to modulate tRNA interactions with the ribosome, explaining the data that showed the importance of nucleotides in positions 32 and 38 for the translational function of tRNAs. One limitation of our experiments thus far is that they rely entirely on tRNA binding or dissociation from the ribosomal A site. The rates that were measured are not a part of normal mechanism of translation and they are 103–104 slower than the rates at which tRNAs enter and leave the A site in the natural process of translation [28,37]. However, recent experiments showed a very good agreement between binding data obtained in a similar manner with both in vitro translation assays and with the Xray structures [38], suggesting that these experiments are relevant for the study of translation. On the other hand, recent studies have shown that in certain buffers, the accuracy of tRNA recognition in translation depends on the specific acceleration of the forward kinetic steps of GTP hydrolysis and peptide bond formation and not on how tight they bind the ribosomes. This is because overall tRNA selection by the ribosome occurs under non-equilibrium conditions [45]. Therefore, it will be important to analyze the role of 32–38 pair identity for tRNA function using assays that measure the tRNA kinetics in translation [46,47].

949

Acknowledgements This work was supported by the National Institutes of Health Grant GM 37552 (to O.C.U.). References [1] P. Auffinger, E. Westhof, Singly and bifurcated hydrogen-bonded basepairs in tRNA anticodon hairpins and ribozymes, J. Mol. Biol. 292 (1999) 467–483. [2] M.I. Baldi, E. Mattoccia, E. Bufardeci, S. Fabbri, G.P. TocchiniValentini, Participation of the intron in the reaction catalyzed by the Xenopus tRNA splicing endonuclease, Science 255 (1992) 1404–1408. [3] P.F. Agris, Decoding the genome: a modified view, Nucleic Acids Res. 32 (2004) 223–238. [4] R. Giege, M. Sissler, C. Florentz, Universal rules and idiosyncratic features in tRNA identity, Nucleic Acids Res. 26 (1998) 5017–5035. [5] M. Sprinzl, C. Horn, M. Brown, A. Ioudovitch, S. Steinberg, Compilation of tRNA sequences and sequences of tRNA genes, Nucleic Acids Res. 26 (1998) 148–153. [6] H. Shi, P.B. Moore, The crystal structure of yeast phenylalanine tRNA at 1.93 Å resolution: a classic structure revisited, RNA 6 (2000) 1091– 1105. [7] E. Westhof, P. Dumas, D. Moras, Restrained refinement of two crystalline forms of yeast aspartic acid and phenylalanine transfer RNA crystals, Acta Crystallogr. A 44 (1988) 112–123. [8] T.L. Bullock, N. Uter, T.A. Nissan, J.J. Perona, Amino acid discrimination by a class I aminoacyl-tRNA synthetase specified by negative determinants, J. Mol. Biol. 328 (2003) 395–408. [9] P. Auffinger, E. Westhof, An extended structural signature for the tRNA anticodon loop, RNA 7 (2001) 334–341. [10] J.M. Ogle, D.E. Brodersen, W.M. Clemons Jr., M.J. Tarry, A.P. Carter, V. Ramakrishnan, Recognition of cognate transfer RNA by the 30S ribosomal subunit, Science 292 (2001) 897–902. [11] P. Nissen, S. Thirup, M. Kjeldgaard, J. Nyborg, The crystal structure of Cys-tRNACys-EF-Tu-GDPNP reveals general and specific features in the ternary complex and in tRNA, Struct. Fold. Des. 7 (1999) 143–156. [12] A. Yaremchuk, M. Tukalo, M. Grotli, S. Cusack, A succession of substrate induced conformational changes ensures the amino acid specificity of Thermus thermophilus prolyl-tRNA synthetase: comparison with histidyl-tRNA synthetase, J. Mol. Biol. 309 (2001) 989–1002. [13] J. Cabello-Villegas, M.E. Winkler, E.P. Nikonowicz, Solution conformations of unmodified and A(37)N(6)-dimethylallyl modified anticodon stem-loops of Escherichia coli tRNA(Phe), J. Mol. Biol. 319 (2002) 1015–1034. [14] M.M. Yusupov, G.Z. Yusupova, A. Baucom, K. Lieberman, T.N. Earnest, J.H. Cate, H.F. Noller, Crystal structure of the ribosome at 5.5 Å resolution, Science 292 (2001) 883–896. [15] A.P. Carter, W.M. Clemons, D.E. Brodersen, R.J. Morgan-Warren, B.T. Wimberly, V. Ramakrishnan, Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics, Nature 407 (2000) 340–348. [16] N.M. Abdi, K. Fredrick, Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli, RNA 11 (2005) 1624–1632. [17] M. Yarus, Translational efficiency of transfer RNA’s: uses of an extended anticodon, Science 218 (1982) 646–652. [18] M. Yarus, S.W. Cline, P. Wier, L. Breeden, R.C. Thompson, Actions of the anticodon arm in translation on the phenotypes of RNA mutants, J. Mol. Biol. 192 (1986) 235–255. [19] C. Marck, H. Grosjean, tRNomics: analysis of tRNA genes from 50 genomes of Eukarya, Archaea, and Bacteria reveals anticodon-sparing strategies and domain-specific features, RNA 8 (2002) 1189–1232. [20] M. Olejniczak, T. Dale, R.P. Fahlman, O.C. Uhlenbeck, Idiosyncratic tuning of tRNAs to achieve uniform ribosome binding, Nat. Struct. Mol. Biol. 12 (2005) 788–793.

950

M. Olejniczak, O.C. Uhlenbeck / Biochimie 88 (2006) 943–950

[21] L.A. Raftery, M. Yarus, Systematic alterations in the anticodon arm make tRNA(Glu)-Suoc a more efficient suppressor, EMBO J. 6 (1987) 1499–1506. [22] M. Yarus, S. Cline, L. Raftery, P. Wier, D. Bradley, The translational efficiency of tRNA is a property of the anticodon arm, J. Biol. Chem. 261 (1986) 10496–10505. [23] Y.M. Hou, P. Schimmel, A simple structural feature is a major determinant of the identity of a transfer RNA, Nature 333 (1988) 140–145. [24] L.G. Kleina, J.M. Masson, J. Normanly, J. Abelson, J.H. Miller, Construction of Escherichia coli amber suppressor tRNA genes. II. Synthesis of additional tRNA genes and improvement of suppressor efficiency, J. Mol. Biol. 213 (1990) 705–717. [25] W.H. McClain, J. Schneider, S. Bhattacharya, K. Gabriel, The importance of tRNA backbone-mediated interactions with synthetase for aminoacylation, Proc. Natl. Acad. Sci. USA 95 (1998) 460–465. [26] F. Tsai, J.F. Curran, tRNA(2Gln) mutants that translate the CGA arginine codon as glutamine in Escherichia coli, RNA 4 (1998) 1514–1522. [27] M. O’Connor, tRNA imbalance promotes -1 frameshifting via nearcognate decoding, J. Mol. Biol. 279 (1998) 727–736. [28] R.P. Fahlman, T. Dale, O.C. Uhlenbeck, Uniform binding of aminoacylated transfer RNAs to the ribosomal A and P sites, Mol. Cell 16 (2004) 799–805. [29] A.D. Wolfson, F.J. LaRiviere, J.A. Pleiss, T. Dale, H. Asahara, O.C. Uhlenbeck, tRNA conformity, Cold Spring Harb. Symp. Quant. Biol. 66 (2001) 185–193. [30] T. Dale, O.C. Uhlenbeck, Amino acid specificity in translation, Trends Biochem. Sci. 30 (2005) 659–665. [31] T. Dale, O.C. Uhlenbeck, Binding of misacylated tRNAs to the ribosomal A site, RNA 11 (2005) 1610–1615. [32] B.H. Mims, N.E. Prather, E.J. Murgola, Isolation and nucleotide sequence analysis of tRNAAlaGGC from Escherichia coli K-12, J. Bacteriol. 162 (1985) 837–839. [33] F. Lustig, T. Boren, Y.S. Guindy, P. Elias, T. Samuelsson, C.W. Gehrke, K.C. Kuo, U. Lagerkvist, Codon discrimination and anticodon structural context, Proc. Natl. Acad. Sci. USA 86 (1989) 6873–6877. [34] F. Lustig, T. Boren, C. Claesson, C. Simonsson, M. Barciszewska, U. Lagerkvist, The nucleotide in position 32 of the tRNA anticodon loop determines ability of anticodon UCC to discriminate among glycine codons, Proc. Natl. Acad. Sci. USA 90 (1993) 3343–3347.

[35] C. Claesson, F. Lustig, T. Boren, C. Simonsson, M. Barciszewska, U. Lagerkvist, Glycine codon discrimination and the nucleotide in position 32 of the anticodon loop, J. Mol. Biol. 247 (1995) 191–196. [36] T. Samuelsson, T. Axberg, T. Boren, U. Lagerkvist, Unconventional reading of the glycine codons, J. Biol. Chem. 258 (1983) 13178–13184. [37] R.P. Fahlman, O.C. Uhlenbeck, Contribution of the esterified amino acid to the binding of aminoacylated tRNAs to the ribosomal P- and A-sites, Biochemistry 43 (2004) 7575–7583. [38] R.P. Fahlman, M. Olejniczak, O.C. Uhlenbeck, Quantitative analysis of deoxynucleotide substitutions in the codon–anticodon helix, J. Mol. Biol. 355 (5) (2006) 887–892. [39] D. Smith, L. Breeden, E. Farrell, M. Yarus, The bases of the tRNA anticodon loop are independent by genetic criteria, Nucleic Acids Res. 15 (1987) 4669–4686. [40] D. Moazed, H.F. Noller, Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16 S rRNA, J. Mol. Biol. 211 (1990) 135–145. [41] J.W. Stuart, Z. Gdaniec, R. Guenther, M. Marszalek, E. Sochacka, A. Malkiewicz, P.F. Agris, Functional anticodon architecture of human tRNALys3 includes disruption of intraloop hydrogen bonding by the naturally occurring amino acid modification, t6A, Biochemistry 39 (2000) 13396–13404. [42] V. Dao, R. Guenther, A. Malkiewicz, B. Nawrot, E. Sochacka, A. Kraszewski, J. Jankowska, K. Everett, P.F. Agris, Ribosome binding of DNA analogs of tRNA requires base modifications and supports the “extended anticodon”, Proc. Natl. Acad. Sci. USA 91 (1994) 2125–2129. [43] J.M. Ogle, F.V. Murphy, M.J. Tarry, V. Ramakrishnan, Selection of tRNA by the ribosome requires a transition from an open to a closed form, Cell 111 (2002) 721–732. [44] S.S. Phelps, S. Joseph, Non-bridging phosphate oxygen atoms within the tRNA anticodon stem-loop are essential for ribosomal A site binding and translocation, J. Mol. Biol. 349 (2005) 288–301. [45] K.B. Gromadski, M.V. Rodnina, Kinetic determinants of high-fidelity tRNA discrimination on the ribosome, Mol. Cell 13 (2004) 191–200. [46] T. Pape, W. Wintermeyer, M.V. Rodnina, Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome, EMBO J. 17 (1998) 7490–7497. [47] L. Cochella, R. Green, An active role for tRNA in decoding beyond codon:anticodon pairing, Science 308 (2005) 1178–1180.