Covariation of a specificity-determining structural motif in an aminoacyl-tRNA synthetase and a tRNA identity element

Transfer RNA (tRNA) identity determinants help preserve the specificity of aminoacylation in vivo, and prevent cross-species interactions. Here, we investigate covariation between the discriminator base (N73) element in histidine tRNAs and residues in the histidyl-tRNA synthetase (HisRS) motif 2 loop. A model of the Escherichia coli HisRS--tRNA(His) complex predicts an interaction between the prokaryotic conserved glutamine 118 of the motif 2 loop and cytosine 73. The substitution of Gln 118 in motif 2 with glutamate decreased discrimination between cytosine and uracil some 50-fold, but left overall rates of adenylation and aminoacylation unaffected. By contrast, substitutions at neighboring Glu 115 and Arg 121 affected both adenylation and aminoacylation, consistent with their predicted involvement in both half-reactions. Additional evidence for the involvement of the motif 2 loop was provided by functional analysis of a hybrid Saccharomyces cerevisiae-- E. coli HisRS possessing the 11 amino acid motif 2 loop of the yeast enzyme. Despite an overall decreased activity of nearly 1000-fold relative to the E. coli enzyme, the chimera nevertheless exhibited a modest preference for the yeast tRNA(His) over the E. coli tRNA, and preferred wild-type yeast tRNA(His) to a variant with C at the discriminator position. These experiments suggest that part of, but not all of, the specificity is provided by the motif 2 loop. The close interaction between enzyme loop and RNA sequence elements suggested by these experiments reflects a covariation between enzyme and tRNA that may have acted to preserve aminoacylation fidelity over evolutionary time.     

G-1:C73 recognition by an arginine cluster in the active site of Escherichia coli histidyl-tRNA synthetase

Aminoacylation of a transfer RNA (tRNA) by its cognate aminoacyl-tRNA synthetase relies upon the recognition of specific nucleotides as well as conformational features within the tRNA by the synthetase. In Escherichia coli, the aminoacylation of tRNA(His) by histidyl-tRNA synthetase (HisRS) is highly dependent upon the recognition of the unique G-1:C73 base pair and the 5'-monophosphate. This work investigates the RNA-protein interactions between the HisRS active site and these critical recognition elements. A homology model of the tRNA(His)-HisRS complex was generated and used to design site-specific mutants of possible G-1:C73 contacts. Aminoacylation assays were performed with these HisRS mutants and N-1:C73 tRNA(His) and microhelix(His) variants. Complete suppression of the negative effect of 5'-phosphate deletion by R123A HisRS, as well as the increased discrimination of Q118E HisRS against a 5'-triphosphate, suggests a possible interaction between the 5'-phosphate and active-site residues Arg123 and Gln118 in which these residues create a sterically and electrostatically favorable pocket for the binding of the negatively charged phosphate group. Additionally, a network of interactions appears likely between G-1 and Arg116, Arg123, and Gln118 because mutation of these residues significantly reduced the sensitivity of HisRS to changes at G-1. Our studies also support an interaction previously proposed between Gln118 and C73. Defining the RNA-protein interactions critical for efficient aminoacylation by E. coli HisRS helps to further characterize the active site of this enzyme and improves our understanding of how the unique identity elements in the acceptor stem of tRNA(His) confer specificity.     

Evolutionary conservation of a functionally important backbone phosphate group critical for aminoacylation of histidine tRNAs

All histidine tRNA molecules have an extra nucleotide, G-1, at the 5' end of the acceptor stem. In bacteria, archaea, and eukaryotic organelles, G-1 base pairs with C73, while in eukaryotic cytoplasmic tRNAHis, G-1 is opposite A73. Previous studies of Escherichia coli histidyl-tRNA synthetase (HisRS) have demonstrated the importance of the G-1:C73 base pair to tRNAHis identity. Specifically, the 5'-monophosphate of G-1 and the major groove amine of C73 are recognized by E. coli HisRS; these individual atomic groups each contribute approximately 4 kcal/mol to transition state stabilization. In this study, two chemically synthesized 24-nucleotide RNA microhelices, each of which recapitulates the acceptor stem of either E. coli or Saccharomyces cervisiae tRNAHis, were used to facilitate an atomic group "mutagenesis" study of the -1:73 base pair recognition by S. cerevisiae HisRS. Compared with E. coli HisRS, microhelixHis is a much poorer substrate relative to full-length tRNAHis for the yeast enzyme. However, the data presented here suggest that, similar to the E. coli system, the 5' monophosphate of yeast tRNA(His) is critical for aminoacylation by yeast HisRS and contributes approximately 3 kcal/mol to transition state stability. The primary role of the unique -1:73 base pair of yeast tRNAHis appears to be to properly position the critical 5' monophosphate for interaction with the yeast enzyme. Our data also suggest that the eukaryotic HisRS/tRNAHis interaction has coevolved to rely less on specific major groove interactions with base atomic groups than the bacterial system.     

Adaptation of aminoacylation identity rules to mammalian mitochondria

Many mammalian mitochondrial aminoacyl-tRNA synthetases are of bacterial-type and share structural domains with homologous bacterial enzymes of the same specificity. Despite this high similarity, synthetases from bacteria are known for their inability to aminoacylate mitochondrial tRNAs, while mitochondrial enzymes do aminoacylate bacterial tRNAs. Here, the reasons for non-aminoacylation by a bacterial enzyme of a mitochondrial tRNA have been explored. A mutagenic analysis performed on in vitro transcribed human mitochondrial tRNA^Asp variants tested for their ability to become aspartylated by Escherichia coli aspartyl-tRNA synthetase, reveals that full conversion cannot be achieved on the basis of the currently established tRNA/synthetase recognition rules. Integration of the full set of aspartylation identity elements and stabilization of the structural tRNA scaffold by restoration of D- and T-loop interactions, enable only a partial gain in aspartylation efficiency. The sequence context and high structural instability of the mitochondrial tRNA are additional features hindering optimal adaptation of the tRNA to the bacterial enzyme. Our data support the hypothesis that non-aminoacylation of mitochondrial tRNAs by bacterial synthetases is linked to the large sequence and structural relaxation of the organelle encoded tRNAs, itself a consequence of the high rate of mitochondrial genome divergence.     

tRNA discrimination at the binding step by a class II aminoacyl-tRNA synthetase.

Aminoacyl-tRNA synthetases preserve the fidelity of decoding genetic information by accurately joining amino acids to their cognate transfer RNAs. Here, tRNA discrimination at the level of binding by Escherichia coli histidyl-tRNA synthetase is addressed by filter binding, analytical ultracentrifugation, and iodine footprinting experiments. Competitive filter binding assays show that the presence of an adenylate analogue 5'-O-[N-(L-histidyl)sulfamoyl]adenosine, HSA, decreased the apparent dissociation constant (K(D)) for cognate tRNA(His) by more than 3-fold (from 3.87 to 1.17 microM), and doubled the apparent K(D) for noncognate tRNA(Phe) (from 7.3 to 14.5 microM). By contrast, no binding discrimination against mutant U73 tRNA(His) was observed, even in the presence of HSA. Additional filter binding studies showed tighter binding of both cognate and noncognate tRNAs by G405D mutant HisRS [Yan, W., Augustine, J., and Francklyn, C. (1996) Biochemistry 35, 6559], which possesses a single amino acid change in the C-terminal anticodon binding domain. Discrimination against noncognate tRNA was also observed in sedimentation velocity experiments, which showed that a stable complex was formed with the cognate tRNA(His) but not with noncognate tRNA(Phe). Footprinting experiments on wild-type versus G405D HisRS revealed characteristic alterations in the pattern of protection and enhancement of iodine cleavage at phosphates 5' to tRNA nucleotides in the anticodon and hinge regions. Together, these results suggest that the anticodon and core regions play major roles in the initial binding discrimination between cognate and noncognate tRNAs, whereas acceptor stem nucleotides, particularly at position 73, influence the reaction at steps after binding of tRNA.     

New computational methods reveal tRNA identity element divergence between Proteobacteria and Cyanobacteria

There are at least 21 subfunctional classes of tRNAs in most cells that, despite a very highly conserved and compact common structure, must interact specifically with different cliques of proteins or cause grave organismal consequences. Protein recognition of specific tRNA substrates is achieved in part through class-restricted tRNA features called tRNA identity determinants. In earlier work we used TFAM, a statistical classifier of tRNA function, to show evidence of unexpectedly large diversity among bacteria in tRNA identity determinants. We also created a data reduction technique called function logos to visualize identity determinants for a given taxon. Here we show evidence that determinants for lysylated isoleucine tRNAs are not the same in Proteobacteria as in other bacterial groups including the Cyanobacteria. Consistent with this, the lysylating biosynthetic enzyme TilS lacks a C-terminal domain in Cyanobacteria that is present in Proteobacteria. We present here, using function logos, a map estimating all potential identity determinants generally operational in Cyanobacteria and Proteobacteria. To further isolate the differences in potential tRNA identity determinants between Proteobacteria and Cyanobacteria, we created two new data reduction visualizations to contrast sequence and function logos between two taxa. One, called Information Difference logos (ID logos), shows the evolutionary gain or retention of functional information associated to features in one lineage. The other, Kullback-Leibler divergence Difference logos (KLD logos), shows recruitments or shifts in the functional associations of features, especially those informative in both lineages. We used these new logos to specifically isolate and visualize the differences in potential tRNA identity determinants between Proteobacteria and Cyanobacteria. Our graphical results point to numerous differences in potential tRNA identity determinants between these groups. Although more differences in general are explained by shifts in functional association rather than gains or losses, the apparent identity differences in lysylated isoleucine tRNAs appear to have evolved through both mechanisms.     

Proteobacterial Histidine-Biosynthetic Pathways Are Paraphyletic

In Lactococcus lactis there is a protein, HisZ, in the histidine-biosynthetic operon that exhibits significant sequence identity with histidyl-tRNA synthetase (HisRS) but does not aminoacylate tRNA. HisRS homologs that, like HisZ, cannot aminoacylate tRNA are represented in a highly divergent set of bacteria (including an aquificale, cyanobacteria, firmicutes, and proteobacteria), yet are missing from other bacteria, including mycrobacteria and certain proteobacteria. Phylogenetic analysis of the HisRS and HisRS-like family suggests that the HisZ proteins form a monophyletic group that attaches outside the predominant bacterial HisRS clade. These observations are consistent with a model in which the absences of HisZ from bacteria are due to its loss during evolution. It has recently been shown that HisZ from L. lactis binds to the ATP-PRPP transferase (HisG) and that both HisZ and HisG are required for catalyzing the first reaction in histidine biosynthesis. Phylogenetic analysis of HisG sequences shows conclusively that proteobacterial HisG and histidinol dehydrogenase (HisD) sequences are paraphyletic and that the partition of the Proteobacteria associated with the presence/absence of HisZ corresponds to that based on HisG and HisD paraphyly. Our results suggest that horizontal gene transfer played an important role in the evolution of the regulation of histidine biosynthesis. Received: 16 July 1999 / Accepted: 4 January 2000     

tRNAHis guanylyltransferase adds G-1 to the 5' end of tRNAHis by recognition of the anticodon, one of several features unexpectedly shared with tRNA synthetases

All eukaryotic tRNA(His) molecules are unique among tRNA species because they require addition of a guanine nucleotide at the -1 position by tRNA(His) guanylyltransferase, encoded in yeast by THG1. This G(-1) residue is both necessary and sufficient for aminoacylation of tRNA by histidyl-tRNA synthetase in vitro and is required for aminoacylation in vivo. Although Thg1 is presumed to be highly specific for tRNA(His) to prevent misacylation of tRNAs, the source of this specificity is unknown. We show here that Thg1 is >10,000-fold more selective for its cognate substrate tRNA(His) than for the noncognate substrate tRNA(Phe). We also demonstrate that the GUG anticodon of tRNA(His) is a crucial Thg1 identity element, since alteration of this anticodon in tRNA(His) completely abrogates Thg1 activity, and the simple introduction of this GUG anticodon to any of three noncognate tRNAs results in significant Thg1 activity. For tRNA(Phe), k(cat)/K(M) is improved by at least 200-fold. Thg1 is the only protein other than aminoacyl-tRNA synthetases that is known to use the anticodon as an identity element to discriminate among tRNA species while acting at a remote site on the tRNA, an unexpected link given the lack of any identifiable sequence similarity between these two families of proteins. Moreover, Thg1 and tRNA synthetases share two other features: They act in close proximity to one another at the top of the tRNA aminoacyl-acceptor stem, and the chemistry of their respective reactions is strikingly similar.     

Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a bipartite tRNA-binding domain

Protein kinase GCN2 regulates translation in amino acid-starved cells by phosphorylating elF2. GCN2 contains a regulatory domain related to histidyl-tRNA synthetase (HisRS) postulated to bind multiple deacylated tRNAs as a general sensor of starvation. In accordance with this model, GCN2 bound several deacylated tRNAs with similar affinities, and aminoacylation of tRNAphe weakened its interaction with GCN2. Unexpectedly, the C-terminal ribosome binding segment of GCN2 (C-term) was required in addition to the HisRS domain for strong tRNA binding. A combined HisRS+ C-term segment bound to the isolated protein kinase (PK) domain in vitro, and tRNA impeded this interaction. An activating mutation (GCN2c-E803V) that weakens PK-C-term association greatly enhanced tRNA binding by GCN2. These results provide strong evidence that tRNA stimulates the GCN2 kinase moiety by preventing an inhibitory interaction with the bipartite tRNA binding domain.     

Hybridization between mitochondrial heavy strand tDNA and expressed light strand tRNA modulates the function of heavy strand tDNA as light strand replication origin

Mitochondrial heavy strand (HS) tDNA codes for tRNAs and frequently functions as the light strand (LS) replication origin (OL). During replication, HS sites remain single-stranded until their LS complement is synthesized, a state prone to hydrolytic deaminations of C → T and A → G, causing genome-wide deamination gradients starting at OLs and proportional to time spent single-stranded. Gradient strength is proportional to OL formation by HS tDNAs. Hypothetically, hybridization between HS tDNA and its expressed complement tRNA should decrease OL activity for LS-, but not HS-encoded tRNAs. Comparisons between primate genomes and between pathogenic and non-pathogenic human polymorphisms both confirm corresponding predictions on OL activity. In primates, strengths of deamination gradients starting at tDNAs functioning as OLs and coding for LS tRNAs decrease proportionally to stabilities of HS tDNA-LS tRNA hybridization; not so for HS tRNAs. Similarly, in mutants of human HS tDNAs coding for LS tRNAs, pathogenic mutants of tDNAs usually not forming OLs form weaker HS tDNA-LS tRNA duplexes than non-pathogenic ones; the opposite is true for tDNAs usually forming OLs. No trend was detected for HS tDNA coding for HS tRNA. tDNA-tRNA hybridization of the modal (most frequent) human tDNA sequence is more stable than of other, rarer non-pathogenic polymorphisms, suggesting similar but weaker mutational effects on tDNA/tRNA functions than in pathogenic mutants. HS tDNA-LS tRNA hybridization appears to compete with OL formation by HS tDNA self-hybridization.     

Sequence analysis of the glutamate tRNA family: evidence for pseudogenes

Human tRNA(CUCGlu) has been isolated by direct hybridization of the tRNA to 28S ribosomal RNA. We now report the isolation of mouse tRNA(CUCGlu) using the same procedure. Partial sequence analysis of the mouse tRNA shows that it is identical to the human tRNA and to a cloned rat tDNA(CUCGlu) sequence. This mouse tRNA(CUCGlu), however, differs by one nucleotide from a previously cloned mouse tDNA(CUCGlu) sequence, suggesting that the tDNA may be a pseudogene. Further evolutionary comparison of these and other glutamate tRNAs and tDNAs has provided evidence to suggest that two other tDNA(Glu) sequences arose by mutation of functional tRNAGlu genes such that their anticodon sequences were converted from one glutamate isoacceptor to the other. These tDNA sequences may also represent pseudogenes.     

Structural basis for recognition of G-1-containing tRNA by histidyl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) play a crucial role in protein translation by linking tRNAs with cognate amino acids. Among all the tRNAs, only tRNA^His bears a guanine base at position -1 (G-1), and it serves as a major recognition element for histidyl-tRNA synthetase (HisRS). Despite strong interests in the histidylation mechanism, the tRNA recognition and aminoacylation details are not fully understood. We herein present the 2.55 Å crystal structure of HisRS complexed with tRNA^His, which reveals that G-1 recognition is principally nonspecific interactions on this base and is made possible by an enlarged binding pocket consisting of conserved glycines. The anticodon triplet makes additional specific contacts with the enzyme but the rest of the loop is flexible. Based on the crystallographic and biochemical studies, we inferred that the uniqueness of histidylation system originates from the enlarged binding pocket (for the extra base G-1) on HisRS absent in other aaRSs, and this structural complementarity between the 5′ extremity of tRNA and enzyme is probably a result of coevolution of both.     

The exchange of the discriminator base A73 for G is alone sufficient to convert human tRNA(Leu) into a serine-acceptor in vitro.

Transfer RNA (tRNA) identify is maintained by the highly specific interaction of a few defined nucleotides or groups of nucleotides, called identity elements, with the cognate aminoacyl-tRNA synthetase, and by nonproductive interactions with the other 19 aminoacyl-tRNA synthetases. Most tRNAs have a set of identity elements in at least two locations, commonly in the anticodon loop or in the acceptor stem, and at the discriminator base position 73. We have used T7 RNA polymerase transcribed tRNAs to demonstrate that the sole replacement of the discriminator base A73 of human tRNA(Leu) with the tRNA(Ser)-specific G generates a complete identity switch to serine acceptance. The reverse experiment, the exchange of G73 in human tRNA(Ser) for the tRNA(Leu-specific A, causes a total loss of serine specificity without creating any leucine acceptance. These results suggest that the discriminator base A73 of human tRNA(Leu) alone protects this tRNA against serylation by seryl-tRNA synthetase. This is the first report of a complete identity switch caused by an exchange of the discriminator base alone.     

A very poorly expressed tRNA(Ser) is highly concentrated together with replication primer initiator tRNA(Met) in the yeast Ty1 virus-like particles.

The analysis of the tRNAs associated to the virus-like particles produced by the Ty1 element revealed the specific packaging of three major tRNA species, in about equal amounts: the replication primer initiator tRNA(Met), the tRNA(Ser)AGA and a tRNA undetected until now as an expressed species in yeast. The latter tRNA is coded by the already described tDNA(Ser)GCT. This tRNA is enriched more than 150 fold in the particles as compared to its content in total cellular tRNA where it represents less than 0.1% (initiator tRNA(Met) and tRNA(Ser)AGA being 11 and 4 fold enriched respectively). This tRNA is the only species coded by the tDNA(Ser)GCT gene which is found in three copies per genome since no other corresponding expressed tRNA could be detected. This gene is thus very poorly expressed. The high concentration of tRNA(Ser)GCU in the particles compared to its very low cellular content led us to consider its possible implication in Ty specific processes.