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.     

Pathology-related substitutions in human mitochondrial tRNA(Ile) reduce precursor 3' end processing efficiency in vitro

The human mitochondrial genome encodes 22 tRNAs interspersed among the two rRNAs and 11 mRNAs, often without spacers, suggesting that tRNAs must be efficiently excised. Numerous maternally transmitted diseases and syndromes arise from mutations in mitochondrial tRNAs, likely due to defect(s) in tRNA metabolism. We have systematically explored the effect of pathogenic mutations on tRNA^Ile precursor 3′ end maturation in vitro by 3′-tRNase. Strikingly, four pathogenic tRNA^Ile mutations reduce 3′-tRNase processing efficiency (V_max / K_M) to ~10-fold below that of wild-type, principally due to lower V_max. The structural impact of mutations was sought by secondary structure probing and wild-type tRNA^Ile precursor was found to fold into a canonical cloverleaf. Among the mutant tRNA^Ile precursors with the greatest 3′ end processing deficiencies, only G4309A displays a secondary structure substantially different from wild-type, with changes in the T domain proximal to the substitution. Reduced efficiency of tRNA^Ile precursor 3′ end processing, in one case associated with structural perturbations, could thus contribute to human mitochondrial diseases caused by mutant tRNAs.     

Sequence-specific recognition of colicin E5, a tRNA-targeting ribonuclease

Colicin E5 is a novel Escherichia coli ribonuclease that specifically cleaves the anticodons of tRNA^Tyr, tRNA^His, tRNA^Asn and tRNA^Asp. Since this activity is confined to its 115 amino acid long C-terminal domain (CRD), the recognition mechanism of E5-CRD is of great interest. The four tRNA substrates share the unique sequence UQU within their anticodon loops, and are cleaved between Q (modified base of G) and 3′ U. Synthetic minihelix RNAs corresponding to the substrate tRNAs were completely susceptible to E5-CRD and were cleaved in the same manner as the authentic tRNAs. The specificity determinant for E5-CRD was YGUN at −1 to +3 of the ‘anticodon’. The YGU is absolutely required and the extent of susceptibility of minihelices depends on N (third letter of the anticodon) in the order A > C > G > U accounting for the order of susceptibility tRNA^Tyr > tRNA^Asp > tRNA^His, tRNA^Asn. Contrastingly, we showed that GpUp is the minimal substrate strictly retaining specificity to E5-CRD. The effect of contiguous nucleotides is inconsistent between the loop and linear RNAs, suggesting that nucleotide extension on each side of GpUp introduces a structural constraint, which is reduced by a specific loop structure formation that includes a 5′ pyrimidine and 3′ A.     

A deafness-associated tRNAHis mutation alters the mitochondrial function,ROS production and membrane potential

In this report, we investigated the molecular genetic mechanism underlying the deafness-associated mitochondrial tRNA^His 12201T>C mutation. The destabilization of a highly conserved base-pairing (5A-68U) by the m.12201T>C mutation alters structure and function of tRNA^His. Using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mtDNA-less (ρ^o) cells, we showed ∼70% decrease in the steady-state level of tRNA^His in mutant cybrids, compared with control cybrids. The mutation changed the conformation of tRNA^His, as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. However, ∼60% increase in aminoacylated level of tRNA^His was observed in mutant cells. The failure in tRNA^His metabolism was responsible for the variable reductions in seven mtDNA-encoded polypeptides in mutant cells, ranging from 37 to 81%, with the average of ∼46% reduction, as compared with those of control cells. The impaired mitochondrial translation caused defects in respiratory capacity in mutant cells. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cells. The data provide the evidence for a mitochondrial tRNA^His mutation leading to deafness.     

Structural Studies of a Bacterial tRNAHIS Guanylyltransferase (Thg1)-Like Protein,with Nucleotide in the Activation and Nucleotidyl Transfer Sites

All nucleotide polymerases and transferases catalyze nucleotide addition in a 5′ to 3′ direction. In contrast, tRNA^His guanylyltransferase (Thg1) enzymes catalyze the unusual reverse addition (3′ to 5′) of nucleotides to polynucleotide substrates. In eukaryotes, Thg1 enzymes use the 3′–5′ addition activity to add G₋₁ to the 5′-end of tRNA^His, a modification required for efficient aminoacylation of the tRNA by the histidyl-tRNA synthetase. Thg1-like proteins (TLPs) are found in Archaea, Bacteria, and mitochondria and are biochemically distinct from their eukaryotic Thg1 counterparts TLPs catalyze 5′-end repair of truncated tRNAs and act on a broad range of tRNA substrates instead of exhibiting strict specificity for tRNA^His. Taken together, these data suggest that TLPs function in distinct biological pathways from the tRNA^His maturation pathway, perhaps in tRNA quality control. Here we present the first crystal structure of a TLP, from the gram-positive soil bacterium Bacillus thuringiensis (BtTLP). The enzyme is a tetramer like human THG1, with which it shares substantial structural similarity. Catalysis of the 3′–5′ reaction with 5′-monophosphorylated tRNA necessitates first an activation step, generating a 5′-adenylylated intermediate prior to a second nucleotidyl transfer step, in which a nucleotide is transferred to the tRNA 5′-end. Consistent with earlier characterization of human THG1, we observed distinct binding sites for the nucleotides involved in these two steps of activation and nucleotidyl transfer. A BtTLP complex with GTP reveals new interactions with the GTP nucleotide in the activation site that were not evident from the previously solved structure. Moreover, the BtTLP-ATP structure allows direct observation of ATP in the activation site for the first time. The BtTLP structural data, combined with kinetic analysis of selected variants, provide new insight into the role of key residues in the activation step.     

Importance of discriminator base stacking interactions: molecular dynamics analysis of A73 microhelix(Ala) variants

Transfer of alanine from Escherichia coli alanyl-tRNA synthetase (AlaRS) to RNA minihelices that mimic the amino acid acceptor stem of tRNA^Ala has been shown, by analysis of variant minihelix aminoacylation activities, to involve a transition state sensitive to changes in the ‘discriminator’ base at position 73. Solution NMR has indicated that this single-stranded nucleotide is predominantly stacked onto G1 of the first base pair of the alanine acceptor stem helix. We report the activity of a new variant with the adenine at position 73 substituted by its non-polar isostere 4-methylindole (M). Despite lacking N7, this analog is well tolerated by AlaRS. Molecular dynamics (MD) simulations show that the M substitution improves position 73 base stacking over G1, as measured by a stacking lifetime analysis. Additional MD simulations of wild-type microhelix^Ala and six variants reveal a positive correlation between N73 base stacking propensity over G1 and aminoacylation activity. For the two ΔN7 variants simulated we found that the propensity to stack over G1 was similar to the analogous variants that contain N7 and we conclude that the decrease in aminoacylation efficiency observed upon deletion of N7 is likely due to loss of a direct stabilizing interaction with the synthetase.     

Plant mitochondria use two pathways for the biogenesis of tRNAHis

All tRNA^His possess an essential extra G_–1 guanosine residue at their 5′ end. In eukaryotes after standard processing by RNase P, G_–1 is added by a tRNA^His guanylyl transferase. In prokaryotes, G_–1 is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G_–1 we find here that both maturation pathways can be used. Indeed, tRNA^His with or without a G_–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNA^His precursors at both positions G₊₁ and G_–1. The G_–1 is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNA^His without G_–1 has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNA^His with a G_–1 are recovered. This shows that a previously unreported tRNA^His guanylyltransferase activity is present in plant mitochondria.     

tRNAHis 5-methylcytidine levels increase in response to several growth arrest conditions in Saccharomyces cerevisiae

tRNAs are highly modified, each with a unique set of modifications. Several reports suggest that tRNAs are hypomodified or, in some cases, hypermodified under different growth conditions and in certain cancers. We previously demonstrated that yeast strains depleted of tRNA^His guanylyltransferase accumulate uncharged tRNA^His lacking the G₋₁ residue and subsequently accumulate additional 5-methylcytidine (m⁵C) at residues C₄₈ and C₅₀ of tRNA^His, due to the activity of the m⁵C-methyltransferase Trm4. We show here that the increase in tRNA^His m⁵C levels does not require loss of Thg1, loss of G₋₁ of tRNA^His, or cell death but is associated with growth arrest following different stress conditions. We find substantially increased tRNA^His m⁵C levels after temperature-sensitive strains are grown at nonpermissive temperature, and after wild-type strains are grown to stationary phase, starved for required amino acids, or treated with rapamycin. We observe more modest accumulations of m⁵C in tRNA^His after starvation for glucose and after starvation for uracil. In virtually all cases examined, the additional m⁵C on tRNA^His occurs while cells are fully viable, and the increase is neither due to the GCN4 pathway, nor to increased Trm4 levels. Moreover, the increased m⁵C appears specific to tRNA^His, as tRNA^Val(AAC) and tRNA^Gly(GCC) have much reduced additional m⁵C during these growth arrest conditions, although they also have C₄₈ and C₅₀ and are capable of having increased m⁵C levels. Thus, tRNA^His m⁵C levels are unusually responsive to yeast growth conditions, although the significance of this additional m⁵C remains unclear.     

Pentatricopeptide repeats of protein-only RNase P use a distinct mode to recognize conserved bases and structural elements of pre-tRNA

Pentatricopeptide repeat (PPR) motifs are α-helical structures known for their modular recognition of single-stranded RNA sequences with each motif in a tandem array binding to a single nucleotide. Protein-only RNase P 1 (PRORP1) in Arabidopsis thaliana is an endoribonuclease that uses its PPR domain to recognize precursor tRNAs (pre-tRNAs) as it catalyzes removal of the 5′-leader sequence from pre-tRNAs with its NYN metallonuclease domain. To gain insight into the mechanism by which PRORP1 recognizes tRNA, we determined a crystal structure of the PPR domain in complex with yeast tRNA^Phe at 2.85 Å resolution. The PPR domain of PRORP1 bound to the structurally conserved elbow of tRNA and recognized conserved structural features of tRNAs using mechanisms that are different from the established single-stranded RNA recognition mode of PPR motifs. The PRORP1 PPR domain-tRNA^Phe structure revealed a conformational change of the PPR domain upon tRNA binding and moreover demonstrated the need for pronounced overall flexibility in the PRORP1 enzyme conformation for substrate recognition and catalysis. The PRORP1 PPR motifs have evolved strategies for protein-tRNA interaction analogous to tRNA recognition by the RNA component of ribonucleoprotein RNase P and other catalytic RNAs, indicating convergence on a common solution for tRNA substrate recognition.     

3′-5′ tRNA guanylyltransferase in bacteria

The identity of the histidine specific transfer RNA (tRNA^His) is largely determined by a unique guanosine residue at position −1. In eukaryotes and archaea, the tRNA^His guanylyltransferase (Thg1) catalyzes 3′-5′ addition of G to the 5′-terminus of tRNA^His. Here, we show that Thg1 also occurs in bacteria. We demonstrate in vitro Thg1 activity for recombinant enzymes from the two bacteria Bacillus thuringiensis and Myxococcus xanthus and provide a closer investigation of several archaeal Thg1. The reaction mechanism of prokaryotic Thg1 differs from eukaryotic enzymes, as it does not require ATP. Complementation of a yeast thg1 knockout strain with bacterial Thg1 verified in vivo activity and suggests a relaxed recognition of the discriminator base in bacteria.     

Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNAHis Pair

While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNA^His have distinctive identity elements, we constructed E. coli tRNA^His _CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNA^His _CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNA^His _CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNA^His _CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.     

Primary structure of three tRNAs from brewer's yeast: tRNA2, tRNA1 and tRNA2

The primary structures of three brewer's yeast tRNAs: tRNA^Pro₂ and tRNA^His_{1 and 2} have been determined

Full-size image (14K)

The U* in the anticodon U*-G-G of tRNA^Pro₂ is probably a derivative of U; tRNA^Pro₂ has 80 per cent homology with mammalian tRNAs^Pro. tRNA^His₁ and tRNA^His₂ differ by only 5 nucleotides; they have identical anticodons and may therefore recognize both codons for histidine; they have an additional nucleotide at the 5′ end. As in all other sequenced tRNAs^His this nucleotide is not paired with the fourth nucleotide from acceptor adenosine. All three sequenced tRNAs have a low degree of homology with their counterparts from yeast mitochondria.