A tRNA identity switch mediated by the binding interaction between a tRNA anticodon and the accessory domain of a class II aminoacyl-tRNA synthetase

Identity elements in tRNAs and the intracellular balance of tRNAs allow accurate selection of tRNAs by aminoacyl-tRNA synthetases. The histidyl-tRNA from Escherichia coli is distinguished by a unique G-1.C73 base pair that upon exchange with other nucleotides leads to a marked decrease in the rate of aminoacylation in vitro. G-1.C73 is also a major identity element for histidine acceptance, such that the substitution of C73 brings about mischarging by glycyl-, glutaminyl-, and leucyl-tRNA synthetases. These identity conversions mediated by the G-1.C73 base pair were exploited to isolate secondary site revertants in the histidyl-tRNA synthetase from E. coli which restore histidine identity to a histidyl-tRNA suppressor carrying U73. The revertant substitutions confer a 3-4 fold reduction in the Michaelis constant for tRNAs carrying the amber-suppressing anticodon and map to the C-terminal domain of HisRS and its interface with the catalytic core. These findings demonstrate that the histidine tRNA anticodon plays a significant role in tRNA selection in vivo and that the C-terminal domain of HisRS is in large part responsible for recognizing this trinucleotide. The kinetic parameters determined also show a small degree of anticooperativity (delta delta G = -1.24 kcal/mol) between recognition of the discriminator base and the anticodon, suggesting that the two helical domains of the tRNA are not recognized independently. We propose that these effects substantially account for the ability of small changes in tRNA binding far removed from the site of a major determinant to bring about a complete conversion of tRNA identity.     

Aspartate identity of transfer RNAs

Structure/function relationships accounting for specific tRNA charging by class II aspartyl-tRNA synthetases from Saccharomyces cerevisiae, Escherichia coli and Thermus thermophilus are reviewed. Effects directly linked to tRNA features are emphasized and aspects about synthetase contribution in expression of tRNA(Asp) identity are also covered. Major identity nucleotides conferring aspartate specificity to yeast, E coli and T thermophilus tRNAs comprise G34, U35, C36, C38 and G73, a set of nucleotides conserved in tRNA(Asp) molecules of other biological origin. Aspartate specificity can be enhanced by negative discrimination preventing, eg mischarging of native yeast tRNA(Asp by yeast arginyl-tRNA synthetase. In the yeast system crystallography shows that identity nucleotides are in contact with identity amino acids located in the catalytic and anticodon binding domains of the synthetase. Specificity of RNA/protein interaction involves a conformational change of the tRNA that optimizes the H-bonding potential of the identity signals on both partners of the complex. Mutation of identity nucleotides leads to decreased aspartylation efficiencies accompanied by a loss of specific H-bonds and an altered adaptation of tRNA on the synthetase. Species-specific characteristics of aspartate systems are the number, location and nature of minor identity signals. These features and the structural variations in aspartate tRNAs and synthetases are correlated with mechanistic differences in the aminoacylation reactions catalyzed by the various aspartyl-tRNA synthetases. The reality of the aspartate identity set is verified by its functional expression in a variety of RNA frameworks. Inversely a number of identities can be expressed within a tRNA(Asp) framework. From this emerged the concept of the RNA structural frameworks underlying expression of identities which is illustrated with data obtained with engineered tRNAs. Efficient aspartylation of minihelices is explained by the primordial role of G73. From this and other considerations it is suggested that aspartate identity appeared early in the history of tRNA aminoacylation systems.     

Identity elements and aminoacylation of plant tRNATrp

Mutation of the Arabidopsis thaliana tRNA (Trp)(CCA) anticodon or of the A73 discriminator base greatly diminishes in vitro aminoacylation with tryptophan, indicating the importance of these nucleotides for recognition by the plant tryptophanyl-tRNA synthetase. Mutation of the tRNA (Trp)(CCA) anticodon to CUA so as to translate amber nonsense codons permits tRNA (Trp)(CCA) to be aminoacylated by A.thaliana lysyl-tRNA synthetase. Thus, translational suppression by tRNA (TRP)(CCA) observed in plant cells includes significant incorporation of lysine into protein.     

Specific atomic groups and RNA helix geometry in acceptor stem recognition by a tRNA synthetase

Oligonucleotides that recapitulate the acceptor stems of tRNAs are substrates for aminoacylation by many tRNA synthetases in vitro, even though these substrates are missing the anticodon trinucleotides of the genetic code. In the case of tRNAAla a single acceptor stem G.U base pair at position 3.70 is essential, based on experiments where the wobble pair has been replaced by alternatives such as I.U, G.C, and A.U, among others. These experiments led to the conclusion that the minor-groove free 2-amino group (of guanosine) of the G.U wobble pair is essential for charging. Moreover, alanine-inserting tRNAs (amber suppressors) that replace G. U with mismatches such as G.A and C.A are partially active in vivo and can support growth of an Escherichia coli tRNAAla knockout strain, leading to the hypothesis that a helix irregularity and nucleotide functionalities are important for recognition. Herein we investigate the charging in vitro of oligonucleotide and full-length tRNA substrates that contain mismatches at the position of the G.U pair. Although most of these substrates have undetectable activity, G.A and C.A variants retain some activity, which is, nevertheless, reduced by at least 100-fold. Thus, the in vivo assays are much less sensitive to large changes in aminoacylation kinetic efficiency of 3.70 variants than is the in vitro assay system. Although these functional data do not clarify all of the details, it is now clear that specific atomic groups are substantially more important in determining kinetic efficiency than is a helical distortion. By implication, the activity of mutant tRNAs measured in the in vivo assays appears to be more dependent on factors other than aminoacylation kinetic efficiency.     

tRNA nucleotide 47: an evolutionary enigma

A previous analysis of tRNA sequences suggested a correlation between the absence of a nucleotide at position 47 (nt 47) in the extra loop and the presence of a U13:G22 base pair in the D-stem. We have evaluated the significance of this correlation by determining the in vivo activity of tRNAs containing either a C13:G22 or a U13:G22 pair in tRNA molecules with or without nt 47. Although this correlation might reflect some malfunction of tRNAs lacking nt 47, but containing the C13:G22, assays of the in vivo suppressor activity showed that this tRNA is actually more active than the tRNA with the features found in the database, i.e., a U13:G22 base pair and no nt 47. Moreover, analogous constructs with a GGC anticodon permitted the growth of an Escherichia coli strain deleted for tRNA(Ala)GGC genes equally well. On the other hand, long-term growth experiments with competing E. coli strains harboring the tRNA lacking nt 47, either with the C13:G22 or the U13:G22 base pair demonstrated that the U13:G22 tRNA overtook the C13:G22 strain even when the starting proportion of strains favored the C13:G22 strain. Thus, the preference for the U13:G22 tRNA lacking nt 47 in the sequence database is most likely due to factors that come into play during extended growth or latency rather than to the ability of the tRNA to engage in protein synthesis.     

Crystal structure of tRNA-guanine transglycosylase: RNA modification by base exchange

tRNA-guanine transglycosylases (TGT) are enzymes involved in the modification of the anticodon of tRNAs specific for Asn, Asp, His and Tyr, leading to the replacement of guanine-34 at the wobble position by the hypermodified base queuine. In prokaryotes TGT catalyzes the exchange of guanine-34 with the queuine (.)precursor 7-aminomethyl-7-deazaguanine (preQ1). The crystal structure of TGT from Zymomonas mobilis was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 19% at 1.85 angstrom resolution. The structure consists of an irregular (beta/alpha)8-barrel with a tightly attached C-terminal zinc-containing subdomain. The packing of the subdomain against the barrel is mediated by an alpha-helix, located close to the C-terminus, which displaces the eighth helix of the barrel. The structure of TGT in complex with preQ1 suggests a binding mode for tRNA where the phosphate backbone interacts with the zinc subdomain and the U33G34U35 sequence is recognized by the barrel. This model for tRNA binding is consistent with a base exchange mechanism involving a covalent tRNA-enzyme intermediate. This structure is the first example of a (beta/alpha)-barrel protein interacting specifically with a nucleic acid.     

Microhelix aminoacylation by a class I tRNA synthetase. Non-conserved base pairs required for specificity

Nucleotides in tRNAs that are conserved among isoacceptors are typically considered as candidates for tRNA synthetase recognition, with less importance attached to non-conserved nucleotides. Although the anticodon is an important contributor to the identity of methionine tRNAs, the class I methionine tRNA synthetase aminoacylates microhelices with high specificity. The microhelix substrates are comprised of as few as the 1st 4 base pairs of the acceptor stems of the elongator and initiator methionine tRNAs. For these two tRNAs, only the central 2:71 and 3:70 base pairs are common to the 1st 4 acceptor stem base pairs. We show here that, although the flanking 4:69 base pair is not conserved, a particular substitution at this position substantially reduces the gel electrophoresis-detected aminoacylation of an acceptor stem substrate that has the conserved 2:71 and 3:70 base pairs. Although the two methionine tRNAs have either U:A or G:C at position 4:69, substitution with C:G reduces charging of 9- or 4-base pair substrates that recreate part or all of the acceptor stem of a methionine tRNA. This effect is sufficient for methionine tRNA synthetase to discriminate between the closely related methionine and isoleucine tRNA acceptor stems. The ability to distinguish G:C and U:A from C:G is contrary to a simple scheme for recognition of atoms in the RNA minor groove.     

Crystal structures of three misacylating mutants of Escherichia coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP

Three previously described mutant Escherichia coli glutaminyl-tRNA synthetase (GlnRS) proteins that incorrectly aminoacylate the amber suppressor derived from tRNATyr (supF) with glutamine were cocrystallized with wild-type tRNAGln and their structures determined. In two of the mutant enzymes studied, Asp235, which contacts base pair G3-C70 in the acceptor stem, has been changed to asparagine in GlnRS7 and to glycine in GlnRS10. These mutations result in changed interactions between Asn235 of GlnRS7 and G3-C70 of the tRNA and an altered water structure between Gly235 of GlnRS10 and base pair G3-C70. These structures suggest how the mutant enzymes can show only small changes in their ability to aminoacylate wild-type cognate tRNA on the one hand and yet show a lack of discrimination against a noncognate U3-A70 base pair on the other. In contrast, the change of Ile129 to Thr in GlnRS15 causes virtually no change in the structure of the complex, and the explanation for its ability to misacylate supF is unclear.     

Aminoacylation of tRNA in the evolution of an aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases catalyze aminoacylation of tRNAs by joining an amino acid to its cognate tRNA. The selection of the cognate tRNA is jointly determined by separate structural domains that examine different regions of the tRNA. The cysteine-tRNA synthetase of Escherichia coli has domains that select for tRNAs containing U73, the GCA anticodon, and a specific tertiary structure at the corner of the tRNA L shape. The E. coli enzyme does not efficiently recognize the yeast or human tRNACys, indicating the evolution of determinants for tRNA aminoacylation from E. coli to yeast to human and the coevolution of synthetase domains that interact with these determinants. By successively modifying the yeast and human tRNACys to ones that are efficiently aminoacylated by the E. coli enzyme, we have identified determinants of the tRNA that are important for aminoacylation but that have diverged in the course of evolution. These determinants provide clues to the divergence of synthetase domains. We propose that the domain for selecting U73 is conserved in evolution. In contrast, we propose that the domain for selecting the corner of the tRNA L shape diverged early, after the separation between E. coli and yeast, while that for selecting the GCA-containing anticodon loop diverged late, after the separation between yeast and human.     

tRNA anticodon recognition and specification within subclass IIb aminoacyl-tRNA synthetases

Subclass IIb aminoacyl-tRNA synthetases (Asn-, Asp- and LysRS) recognize the anticodon triplet of their cognate tRNA (GUU, GUC and UUU, respectively) through an OB-folded N-terminal extension. In the present study, the specificity of constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli was analyzed by cross-mutagenesis of the tRNA(Lys) anticodon, on the one hand, and of the amino acid residues composing the anticodon binding site on the other. From this analysis, a tentative model is deduced for both the recognition of the cognate anticodon and the rejection of non-cognate anticodons. In this model, the enzyme offers a rigid scaffold of amino acid residues along the beta-strands of the OB-fold for tRNA binding. Phe85 and Gln96 play a critical role in this spatial organization. This scaffold can recognize directly U35 at the center of the anticodon. Specification of the correct enzyme:tRNA complex is further achieved through the accommodation of U34 and U36. The binding of these bases triggers the conformationnal change of a flexible seven-residue loop between strands 4 and 5 of the OB-fold (L45). Additional free energy of binding is recovered from the resulting network of cooperative interactions. Such a mechanism would not depend on the modifications of the anticodon loop of tRNA(Lys) (mnm5s2U34 and t6A37). In the model, exclusion by the synthetase of non-cognate anticodons can be accounted for by a hindrance to the positioning of the L45 loop. In addition, Glu135 would repulse a cytosine base at position 35. Sequence comparisons show that the composition and length of the L45 loop are markedly conserved in each of the families composing subclass IIb aminoacyl-tRNA synthetases. The possible role of the loop is discussed for each case, including that of archaebacterial aspartyl-tRNA synthetases.     

Aminoacylation of hypomodified tRNAGlu in vivo

The highly specific interaction of each aminoacyl-tRNA synthetase and its substrate tRNAs constitutes an intriguing problem in protein-RNA recognition. All tRNAs have the same overall three-dimensional structure in order to fit interchangeably into the translational apparatus. Thus, the recognition by aminoacyl-tRNA synthetase must be more or less limited to discrimination between bases at specific positions within the tRNA. The hypermodified nucleotide 5-methylaminomethyl-2-thiouridine (mnm5s2U) present at the wobble position of bacterial tRNAs specific for glutamic acid, lysine and possibly glutamine has been shown to be important in the recognition of these tRNAs by their synthetases in vitro. Here, we have determined the aminoacylation level in vivo of tRNAGlu, tRNALys, and tRNA1GIn in Escherichia coli strains containing undermodified derivatives of mnm5s2U34. Lack of the 5-methylaminomethyl group did not reduce charging levels for any of the three tRNAs. Lack of the s2U34 modification caused a 40% reduction in the charging level of tRNAGlu. Charging of tRNALys and tRNA1Gln were less affected. There was no compensating regulation of expression of glutamyl-tRNA synthetase because the relative synthesis rate was the same in the wild-type and mutant strains. These results indicate that the mnm5U34 modification is not an important recognition element in vivo for the glutamyl-tRNA synthetase. In contrast, lack of the s2U34 modification reduced the efficiency of charging by at least 40%. This is the minimal estimate because the turn-over rate of Glu-tRNAGlu was also reduced in the absence of the 2-thio group. Lack of either modification did not affect mischarging or mistranslation.     

Species-specific differences in the operational RNA code for aminoacylation of tRNAPro

An operational RNA code relates amino acids to specific structural features located in tRNA acceptor stems. In contrast to the universal nature of the genetic code, the operational RNA code can vary in evolution due to coadaptations of the contacts between aminoacyl-tRNA synthetases and the acceptor stems of their cognate tRNA substrates. Here we demonstrate that, for class II prolyl-tRNA synthetase (ProRS), functional coadaptations have occurred in going from the bacterial to the human enzyme. Analysis of 20 ProRS sequences that cover all three taxonomic domains (bacteria, eucarya, and archaea) revealed that the sequences are divided into two evolutionarily distant groups. Aminoacylation assays showed that, while anticodon recognition has been maintained through evolution, significant changes in acceptor stem recognition have occurred. Whereas all tRNAPro sequences from bacteria strictly conserve A73 and C1.G72, all available cytoplasmic eukaryotic tRNAPro sequences have a C73 and a G1.C72 base pair. In contrast to the Escherichia coli synthetase, the human enzyme does not use these elements as major recognition determinants, since mutations at these positions have only small effects on cognate synthetase charging. Additionally, E. coli tRNAPro is a poor substrate for human ProRS, and the presence of the human anticodon-D stem biloop domain was necessary and sufficient to confer efficient aminoacylation by human ProRS on a chimeric tRNAPro containing the E. coli acceptor-TpsiC stem-loop domain. Our data suggest that the two ProRS groups may reflect coadaptations needed to accommodate changes in the operational RNA code for proline.