Acquisition of an insertion peptide for efficient aminoacylation by a halophile tRNA synthetase

Enzymes of halophilic organisms contain unusual peptide motifs that are absent from their mesophilic counterparts. The functions of these halophile-specific peptides are largely unknown. Here we have identified an unusual peptide that is unique to several halophile archaeal cysteinyl-tRNA synthetases (CysRS), which catalyze attachment of cysteine to tRNA(Cys) to generate the essential cysteinyl-tRNA(Cys) required for protein synthesis. This peptide is located near the active site in the catalytic domain and is highly enriched with acidic residues. In the CysRS of the extreme halophile Halobacterium species NRC-1, deletion of the peptide reduces the catalytic efficiency of aminoacylation by a factor of 100 that largely results from a defect in kcat, rather than the Km for tRNA(Cys). In contrast, maintaining the peptide length but substituting acidic residues in the peptide with neutral or basic residues has no major deleterious effect, suggesting that the acidity of the peptide is not important for the kcat of tRNA aminoacylation. Analysis of general protein structure under physiological high salt concentrations, by circular dichroism and by fluorescence titration of tRNA binding, indicates little change due to deletion of the peptide. However, the presence of the peptide confers tolerance to lower salt levels, and fluorescence analysis in 30% sucrose reveals instability of the enzyme without the peptide. We suggest that the stability associated with the peptide can be used to promote proper enzyme conformation transitions in various stages of tRNA aminoacylation that are associated with catalysis. The acquisition of the peptide by the halophilic CysRS suggests an enzyme adaptation to high salinity.     

Alternative design of a tRNA core for aminoacylation

The core of Escherichia coli tRNA(Cys) is important for aminoacylation of the tRNA by cysteine-tRNA synthetase. This core differs from the common tRNA core by having a G15:G48, rather than a G15:C48 base-pair. Substitution of G15:G48 with G15:C48 decreases the catalytic efficiency of aminoacylation by two orders of magnitude. This indicates that the design of the core is not compatible with G15:C48. However, the core of E. coli tRNA(Gln), which contains G15:C48, is functional for cysteine-tRNA synthetase. Here, guided by the core of E. coli tRNA(Gln), we sought to test and identify alternative functional design of the tRNA(Cys) core that contains G15:C48. Although analysis of the crystal structure of tRNA(Cys) and tRNA(Gln) implicated long-range tertiary base-pairs above and below G15:G48 as important for a functional core, we showed that this was not the case. The replacement of tertiary interactions involving 9, 21, and 59 in tRNA(Cys) with those in tRNA(Gln) did not construct a functional core that contained G15:C48. In contrast, substitution of nucleotides in the variable loop adjacent to 48 of the 15:48 base-pair created functional cores. Modeling studies of a functional core suggests that the re-constructed core arose from enhanced stacking interactions that compensated for the disruption caused by the G15:C48 base-pair. The repacked tRNA core displayed features that were distinct from those of the wild-type and provided evidence that stacking interactions are alternative means than long-range tertiary base-pairs to a functional core for aminoacylation.     

Influence of transfer RNA tertiary structure on aminoacylation efficiency by glutaminyl and cysteinyl-tRNA synthetases

The position of the tertiary Levitt pair between nucleotides 15 and 48 in the transfer RNA core region suggests a key role in stabilizing the joining of the two helical domains, and in maintaining the relative orientations of the D and variable loops. E. coli tRNA(Gln) possesses the canonical Pu15-Py48 trans pairing at this position (G15-C48), while the tRNA(Cys) species from this organism instead features an unusual G15-G48 pair. To explore the structural context dependence of a G15-G48 Levitt pair, a number of tRNA(Gln) species containing G15-G48 were constructed and evaluated as substrates for glutaminyl and cysteinyl-tRNA synthetases. The glutaminylation efficiencies of these mutant tRNAs are reduced by two to tenfold compared with native tRNA(Gln), consistent with previous findings that the tertiary core of this tRNA plays a role in GlnRS recognition. Introduction of tRNA(Cys) identity nucleotides at the acceptor and anticodon ends of tRNA(Gln) produced a tRNA substrate which was efficiently aminoacylated by CysRS, even though the tertiary core region of this species contains the tRNA(Gln) G15-C48 pair. Surprisingly, introduction of G15-G48 into the non-cognate tRNA(Gln) tertiary core then significantly impairs CysRS recognition. By contrast, previous work has shown that CysRS aminoacylates tRNA(Cys) core regions containing G15-G48 with much better efficiency than those with G15-C48. Therefore, tertiary nucleotides surrounding the Levitt pair must significantly modulate the efficiency of aminoacylation by CysRS. To explore the detailed nature of the structural interdependence, crystal structures of two tRNA(Gln) mutants containing G15-G48 were determined bound to GlnRS. These structures show that the larger purine ring of G48 is accommodated by rotation into the syn position, with the N7 nitrogen serving as hydrogen bond acceptor from several groups of G15. The G15-G48 conformations differ significantly compared to that observed in the native tRNA(Cys) structure bound to EF-Tu, further implicating an important role for surrounding nucleotides in maintaining the integrity of the tertiary core and its consequent ability to present crucial recognition determinants to aminoacyl-tRNA synthetases.     

Stimulatory effect of ammonium sulfate at high concentrations on the aminoacylation of tRNA and tRNA-like molecules

The influence of various salts on the aminoacylation of tRNA(Val) and the tRNA-like structure from turnip yellow mosaic virus RNA by yeast valyl-tRNA synthetase has been studied. As expected, increasing the concentration of salts inhibits the enzymatic reaction. However, in the presence of high concentration of ammonium sulfate, and only this salt, the inhibitory effect is suppressed. Under such conditions, the aminoacylation becomes comparable to that measured in the absence of salt. It was shown that ammonium sulfate affects both the catalytic rate of the reaction and the affinity between valyl-tRNA synthetase and the RNAs. Because the affinity between the partners in the complex is increased when the concentration of the salt is high, it is suggested that hydrophobic effects are involved in tRNA/synthetase interactions.     

Conservation of a tRNA core for aminoacylation

The core region of Escherichia coli tRNA(Cys)is important for aminoacylation of the tRNA. This core contains an unusual G15:G48 base pair, and three adenosine nucleotides A13, A22 and A46 that are likely to form a 46:[13:22] adenosine base triple. We recently observed that the 15:48 base pair and the proposed 46:[13:22] triple are structurally and functionally coupled to contribute to aminoacylation. Inspection of a database of tRNA sequences shows that these elements are only found in one other tRNA, the Haemophilus influenzae tRNA(Cys). Because of the complexity of the core, conservation of sequence does not mean conservation of function. We here tested whether the conserved elements in H. influenzae tRNA(Cys)were also important for aminoacylation of H. influenzae tRNA(Cys). We cloned and purified a recombinant H. influenzae cysteine-tRNA synthe-tase and showed that it depends on 15:48 and 13, 22 and 46 in a relationship analogous to that of E. coli cysteine-tRNA synthetase. The functional conservation of the tRNA core is correlated with sequence conservation between E.coli and H.influenzae cysteine-tRNA synthetases. As the genome of H. influenzae is one of the smallest and may approximate a small autonomous entity in the development of life, the dependence of this genome on G15:G48 and its coupling with the proposed A46:[A13:A22] triple for aminoacylation with cysteine suggests an early role of these motifs in the evolution of decoding genetic information.     

Recognition of tRNA backbone for aminoacylation with cysteine: evolution from Escherichia coli to human

The underlying basis of the genetic code is specific aminoacylation of tRNAs by aminoacyl-tRNA synthetases. Although the code is conserved, bases in tRNA that establish aminoacylation are not necessarily conserved. Even when the bases are conserved, positions of backbone groups that contribute to aminoacylation may vary. We show here that, although the Escherichia coli and human cysteinyl-tRNA synthetases both recognize the same bases (U73 and the GCA anticodon) of tRNA for aminoacylation, they have different emphasis on the tRNA backbone. The E. coli enzyme recognizes two clusters of phosphate groups. One is at A36 in the anticodon and the other is in the core of the tRNA structure and includes phosphate groups at positions 9, 12, 14, and 60. Metal-ion rescue experiments show that those at positions 9, 12, and 60 are involved with binding divalent metal ions that are important for aminoacylation. The E. coli enzyme also recognizes 2'-hydroxyl groups within the same two clusters: at positions 33, 35, and 36 in the anticodon loop, and at positions 49, 55, and 61 in the core. The human enzyme, by contrast, recognizes few phosphate or 2'-hydroxy groups for aminoacylation. The evolution from the backbone-dependent recognition by the E. coli enzyme to the backbone-independent recognition by the human enzyme demonstrates a previously unrecognized shift that nonetheless has preserved the specificity for aminoacylation with cysteine.     

Shape-selective RNA recognition by cysteinyl-tRNA synthetase

The crystal structure of Escherichia coli cysteinyl-tRNA synthetase (CysRS) bound to tRNA(Cys) at a resolution of 2.3 A reveals base-specific and shape-selective interactions across an extensive protein-RNA recognition interface. The complex contains a mixed alpha/beta C-terminal domain, which is disordered in the unliganded enzyme. This domain makes specific hydrogen bonding interactions with all three bases of the GCA anticodon. The tRNA anticodon stem is bent sharply toward the enzyme as compared with its conformation when bound to elongation factor Tu, providing an essential basis for shape-selective recognition. The CysRS structure also reveals interactions of conserved enzyme groups with the sugar-phosphate backbone in the D loop, adjacent to an unusual G15.G48 tertiary base pair previously implicated in tRNA aminoacylation. A combined mutational analysis of enzyme and tRNA groups at G15.G48 supports the notion that contacts between CysRS and the sugar-phosphate backbone contribute to recognition by indirect readout.     

Kinetic quality control of anticodon recognition by a eukaryotic aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases are an ancient class of enzymes responsible for the matching of amino acids with anticodon sequences of tRNAs. Eukaryotic tRNA synthetases are often larger than their bacterial counterparts, and several mammalian enzymes use the additional domains to facilitate assembly into a multi-synthetase complex. Human cysteinyl-tRNA synthetase (CysRS) does not associate with the multi-synthetase complex, yet contains a eukaryotic-specific C-terminal extension that follows the tRNA anticodon-binding domain. Here we show by mutational and kinetic analysis that the C-terminal extension of human CysRS is used to selectively improve recognition and binding of the anticodon sequence, such that the specificity of anticodon recognition by human CysRS is higher than that of its bacterial counterparts. However, the improved anticodon recognition is achieved at the expense of a significantly slower rate in the aminoacylation reaction, suggesting a previously unrecognized kinetic quality control mechanism. This kinetic quality control reflects an evolutionary adaptation of some tRNA synthetases to improve the anticodon specificity of tRNA aminoacylation from bacteria to humans, possibly to accommodate concomitant changes in codon usage.     

Amino acid discrimination by a highly differentiated metal center of an aminoacyl-tRNA synthetase

Escherichia coli cysteinyl-tRNA synthetase (CysRS) achieves high amino acid specificity without the need for an editing reaction. Crystallographic and spectroscopic studies have previously demonstrated that a major determinant of the specificity is an active site zinc ion that recognizes the substrate cysteine through a strong zinc-thiolate interaction. The active site cleft of CysRS is composed of highly or strictly conserved amino acids, including four inner-sphere zinc ligands, five histidine imidazoles at the base of the cleft, and a tryptophan that flips down upon cysteine binding to complete formation of the binding pocket. Here we establish the significance of each of these major features of the active site cleft by mutational analysis. Substitutions generally lead to substantially deleterious effects on K(m) and k(cat) parameters with respect to each of the cysteine, ATP, and tRNA(Cys) substrates. These findings emphasize the importance of the highly differentiated nature of the active site and provide new insights into the origins of selectivity without editing. Most mutants are less attenuated in tRNA aminoacylation than in adenylate synthesis, suggesting that tRNA binding drives a conformational change to help assemble the active site.     

Domain-domain communication for tRNA aminoacylation: the importance of covalent connectivity

Aminoacyl-tRNA synthetases form complexes with tRNA to catalyze transfer of activated amino acids to the 3' end of tRNA. The tRNA synthetase complexes are roughly divided into the activation and tRNA-binding domains of synthetases, which interact with the acceptor and anticodon ends of tRNAs, respectively. Efficient aminoacylation of tRNA by Escherichia coli cysteinyl-tRNA synthetase (CysRS) requires both domains, although the pathways for the long-range domain-domain communication are not well understood. Previous studies show that dissection of tRNA(Cys) into acceptor and anticodon helices seriously reduces the efficiency of aminoacylation, suggesting that communication requires covalent continuity of the tRNA backbone. Here we tested if communication requires the continuity of the synthetase backbone. Two N-terminal fragments and one C-terminal fragment of E. coli CysRS were generated. While the N-terminal fragments were active in adenylate synthesis, they were severely defective in the catalytic efficiency and specificity of tRNA aminoacylation. Conversely, although the C-terminal fragment was not catalytically active, it was able to bind and discriminate tRNA. However, addition of the C-terminal fragment to an N-terminal fragment in trans did not improve the aminoacylation efficiency of the N-terminal fragment to the level of the full-length enzyme. These results emphasize the importance of covalent continuity of both CysRS and tRNA(Cys) for efficient tRNA aminoacylation, and highlight the energetic costs of constraining the tRNA synthetase complex for domain-domain communication. Importantly, this study also provides new insights into the existence of several natural "split" synthetases that are now identified from genomic sequencing projects.     

Evidence for a conserved relationship between an acceptor stem and a tRNA for aminoacylation.

The anticodon-independent aminoacylation of RNA hairpin helices that reconstruct tRNA acceptor stems has been demonstrated for at least 10 aminoacyl-tRNA synthetases. For Escherichia coli cysteine tRNA synthetase, the specificity of aminoacylation of the acceptor stem is determined by the U73 nucleotide adjacent to the amino acid attachment site. Because U73 is present in all known cysteine tRNAs, we investigated the ability of the E. coli cystein enzyme to aminoacylate a heterologous acceptor stem. We show here that a minihelixCys based on the acceptor-T psi C stem of yeast tRNACys is a substrate for the E. coli enzyme, and that aminoacylation of this minihelix is dependent on U73. Additionally, we identify two base pairs in the acceptor stem that quantitatively convert the E. coli acceptor stem to the yeast acceptor stem. The influence of U73 and these two base pairs is completely retained in the full-length tRNA. This suggests a conserved relationship between the acceptor stem alone and the acceptor stem in the context of a tRNA for aminoacylation with cysteine. However, the primary determinant in the species-specific aminoacylation of the E. coli and yeast cysteine tRNAs is a tertiary base pair at position 15:48 outside of the acceptor stem. Although E. coli tRNACys has an unusual G15:G48 tertiary base pair, yeast tRNACys has a more common G15:C48 that prevents efficient aminoacylation of yeast tRNACys by the E. coli enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases

Here we describe the many applications of acid urea polyacrylamide gel electrophoresis (acid urea PAGE) followed by Northern blot analysis to studies of tRNAs and aminoacyl-tRNA synthetases. Acid urea PAGE allows the electrophoretic separation of different forms of a tRNA, discriminated by changes in bulk, charge, and/or conformation that are brought about by aminoacylation, formylation, or modification of a tRNA. Among the examples described are (i) analysis of the effect of mutations in the Escherichia coli initiator tRNA on its aminoacylation and formylation; (ii) evidence of orthogonality of suppressor tRNAs in mammalian cells and yeast; (iii) analysis of aminoacylation specificity of an archaeal prolyl-tRNA synthetase that can aminoacylate archaeal tRNA(Pro) with cysteine, but does not aminoacylate archaeal tRNA(Cys) with cysteine; (iv) identification and characterization of the AUA-decoding minor tRNA(Ile) in archaea; and (v) evidence that the archaeal minor tRNA(Ile) contains a modified base in the wobble position different from lysidine found in the corresponding eubacterial tRNA.     

Indirect readout of tRNA for aminoacylation

Aminoacylation of tRNA by aminoacyl-tRNA synthetases is the essential reaction that matches protein amino acids with the trinucleotide sequences specified in mRNA. Direct electrostatic interactions made by tRNA synthetases with discriminating functional groups on the tRNA bases have long been known to determine aminoacylation specificity. However, structural and biochemical studies have revealed a second "indirect readout" mechanism that makes an important contribution as well. In indirect readout, the sequence-dependent conformations of tRNA are recognized through protein contacts with the sugar-phosphate backbone and with nonspecific portions of the bases. This mechanism appears to function in single-stranded regions, in canonical A-type duplex segments, and in the complex tertiary core portion of the tRNA. Operation of the indirect mechanism is not exclusive of the direct mechanism, and both are further mediated by induced-fit rearrangements, in which enzyme and tRNA undergo precise conformational changes after formation of an initial encounter complex. The examples of indirect readout in tRNA synthetase complexes extend the concept beyond its traditional application to DNA duplexes and serve as models for the operation of this mechanism in more complex systems such as the ribosome.     

Recognition of tRNALeu by Aquifex aeolicus leucyl-tRNA synthetase during the aminoacylation and editing steps

Recognition of tRNA by the cognate aminoacyl-tRNA synthetase during translation is crucial to ensure the correct expression of the genetic code. To understand tRNA^Leu recognition sets and their evolution, the recognition of tRNA^Leu by the leucyl-tRNA synthetase (LeuRS) from the primitive hyperthermophilic bacterium Aquifex aeolicus was studied by RNA probing and mutagenesis. The results show that the base A73; the core structure of tRNA formed by the tertiary interactions U8–A14, G18–U55 and G19–C56; and the orientation of the variable arm are critical elements for tRNA^Leu aminoacylation. Although dispensable for aminoacylation, the anticodon arm carries discrete editing determinants that are required for stabilizing the conformation of the post-transfer editing state and for promoting translocation of the tRNA acceptor arm from the synthetic to the editing site.     

tRNA discrimination by T. thermophilus phenylalanyl-tRNA synthetase at the binding step

The extent of tRNA recognition at the level of binding by Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS), one of the most complex class II synthetases, has been studied by independent measurements of the enzyme association with wild-type and mutant tRNA(Phe)s as well as with non-cognate tRNAs. The data obtained, combined with kinetic data on aminoacylation, clearly show that PheRS exhibits more tRNA selectivity at the level of binding than at the level of catalysis. The anticodon nucleotides involved in base-specific interactions with the enzyme prevail both in the initial binding recognition and in favouring aminoacylation catalysis. Tertiary nucleotides of base pair G19-C56 and base triple U45-G10-C25 contribute primarily to stabilization of the correctly folded tRNA(Phe) structure, which is important for binding. Other nucleotides of the central core (U20, U16 and of the A26-G44 tertiary base pair) are involved in conformational adjustment of the tRNA upon its interaction with the enzyme. The specificity of nucleotide A73, mutation of which slightly reduces the catalytic rate of aminoacylation, is not displayed at the binding step. A few backbone-mediated contacts of PheRS with the acceptor and anticodon stems revealed in the crystal structure do not contribute to tRNA(Phe) discrimination, their role being limited to stabilization of the complex. The highest affinity of T. thermophilus PheRS for cognate tRNA, observed for synthetase-tRNA complexes, results in 100-3000-fold binding discrimination against non-cognate tRNAs.