Characterization of yeast seryl-tRNA synthetase active site mutants with improved discrimination against substrate analogues.

The involvement of amino acids within the motif 2 loop of Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) in serine and ATP binding was demonstrated previously [B. Lenhard et al., J. Biol. Chem. 272 (1997) 1136-1141]. In our attempt to analyze the structural basis for the substrate specificity and to explore further the catalytic mechanism employed by S. cerevisiae SerRS, two new active site mutants, SerRS11 and SerRS12, were constructed. The catalytic effects of amino acid replacement at positions Lys287, Asp288 and Ala289 with purified wild-type and mutant seryl-tRNA synthetases were tested. The alteration of these semi-conserved amino acids interferes with tRNA-dependent optimization of serine recognition. Additionally, mutated enzymes SerRS11 (Lys287Thr, Asp288Tyr, Ala289Val) and SerRS12 (Lys287Arg) are less sensitive to inhibition by two competitive inhibitors: serine hydroxamate, an analogue of serine, and 5'-O-[N-(L-seryl)-sulfamoyl]adenosine, a stable analogue of aminoacyl adenylate, than the wild-type enzyme. SerRS mutants also display different activation kinetics for serine and serine hydroxamate, indicating that specificity toward the substrates is modulated by amino acid replacement in the motif 2 loop.

tRNA recognition and evolution of determinants in seryl-tRNA synthesis.

We have analyzed the evolution of recognition of tRNAsSerby seryl-tRNA synthetases, and compared it to other type 2 tRNAs, which contain a long extra arm. In Eubacteria and chloroplasts this type of tRNA is restricted to three families: tRNALeu, tRNASer and tRNATyr. tRNALeuand tRNASer also carry a long extra arm in Archaea, Eukarya and all organelles with the exception of animal mitochondria. In contrast, the long extra arm of tRNATyr is far less conserved: it was drastically shortened after the separation of Archaea and Eukarya from Eubacteria, and it is also truncated in animal mitochondria. The high degree of phylo-genetic divergence in the length of tRNA variable arms, which are recognized by both class I and class II aminoacyl-tRNA synthetases, makes type 2 tRNA recognition an ideal system with which to study how tRNA discrimination may have evolved in tandem with the evolution of other components of the translation machinery.

C-terminal truncation of yeast SerRS is toxic for Saccharomyces cerevisiae due to altered mechanism of substrate recognition.

Like all other eukaryal cytosolic seryl-tRNA synthetase (SerRS) enzymes, Saccharomyces cerevisiae SerRS contains a C-terminal extension not found in the enzymes of eubacterial and archaeal origin. Overexpression of C-terminally truncated SerRS lacking the 20-amino acid appended domain (SerRSC20) is toxic to S. cerevisiae possibly because of altered substrate recognition. Compared to wild-type SerRS the truncated enzyme displays impaired tRNA-dependent serine recognition and is less stable. This suggests that the C-terminal peptide is important for the formation or maintenance of the enzyme structure optimal for substrate binding and catalysis.

Maize mitochondrial seryl-tRNA synthetase recognizes Escherichia coli tRNA(Ser) in vivo and in vitro.

In our studies to analyze the structure/function relationships among cytoplasmic and organellar seryl-tRNA synthetases (SerRS), we have characterized a Zea mays cDNA (SerZMm) encoding a protein with significant similarity to prokaryotic SerRS enzymes. To demonstrate the functional identity of SerZMm, the gene sequence encoding the putative mature protein was cloned. This construct complemented in vivo a temperature-sensitive Escherichia coli serS mutant strain. The mature SerZMm protein overexpressed in Escherichia coli efficiently aminoacylated bacterial tRNA(Ser) in vitro, while yeast tRNA was a poor substrate. These data identify SerZMm as an organellar maize seryl-tRNA synthetase, the first plant organellar SerRS to be cloned. The analysis of its N-terminal targeting signal suggests a mitochondrial function for the SerZMm protein in maize.

Matrix-assisted laser desorption/ionisation mass spectrometry of transfer ribonucleic acids isolated from yeast.

tRNATyr and tRNASer purified from bulk brewer's yeast tRNA were subjected to analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Choosing a mixture of 2,4,6- and 2,3,4-trihydroxy-acetophenone and diammonium citrate as matrix a mass resolution of up to 220 (FWHM) was achieved in the linear mode of operation. Cation adduct suppression by addition of cation exchange beads and a chelating agent (CDTA) is shown to substantially improve mass resolution for this class of molecules.

Defining the active site of yeast seryl-tRNA synthetase. Mutations in motif 2 loop residues affect tRNA-dependent amino acid recognition.

The active site of class II aminoacyl-tRNA synthetases contains the motif 2 loop, which is involved in binding of ATP, amino acid, and the acceptor end of tRNA. In order to characterize the active site of Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS), we performed in vitro mutagenesis of the portion of the SES1 gene encoding the motif 2 loop. Substitutions of amino acids conserved in the motif 2 loop of seryl-tRNA synthetases from other sources led to loss of complementation of a yeast SES1 null allele strain by the mutant yeast SES1 genes. Steady-state kinetic analyses of the purified mutant SerRS proteins revealed elevated Km values for serine and ATP, accompanied by decreases in kcat (as expected for replacement of residues involved in aminoacyl-adenylate formation). The differences in the affinities for serine and ATP, in the absence and presence of tRNA are consistent with the proposed conformational changes induced by positioning the 3'-end of tRNA into the active site, as observed recently in structural studies of Thermus thermophilus SerRS (Cusack, S., Yaremchuk, A., and Tukalo, M. (1996) EMBO J. 15, 2834-2842). The crystal structure of this moderately homologous prokaryotic counterpart of the yeast enzyme allowed us to produce a model of the yeast SerRS structure and to place the mutations in a structural context. In conjunction with structural data for T. thermophilus SerRS, the kinetic data presented here suggest that yeast seryl-tRNA synthetase displays tRNA-dependent amino acid recognition.

Transfer RNA-dependent cognate amino acid recognition by an aminoacyl-tRNA synthetase.

An investigation of the role of tRNA in the catalysis of aminoacylation of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) has revealed that the accuracy of specific interactions between GlnRS and tRNAGln determines amino acid affinity. Mutations in GlnRS at D235, which makes contacts with nucleotides in the acceptor stem of tRNAGln, and at R260 in the enzyme's active site were found to be independent during tRNA binding but interactive for aminoacylation. Characterization of mutants of GlnRS at position 235, showed amino acid recognition to be tRNA mediated. Aminoacylation of tRNA(CUA)Tyr [tyrT (UAG)] by GlnRS-D235H resulted in a 4-fold increase in the Km for the Gln, which was reduced to a 2-fold increase when A73 was replaced with G73. These and previous results suggest that specific interactions between GlnRS and tRNAGln ensure the accurate positioning of the 3' terminus. Disruption of these interactions can change the Km for Gln over a 30-fold range, indicating that the accuracy of aminoacylation is regulated by tRNA at the level of both substrate recognition and catalysis. The observed role of RNA as a cofactor in optimizing amino acid activation suggests that the tRNAGln-GlnRS complex may be partly analogous to ribonucleoprotein enzymes where protein-RNA interactions facilitate catalysis.

The C-terminal extension of yeast seryl-tRNA synthetase affects stability of the enzyme and its substrate affinity.

Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) contains a 20-amino acid C-terminal extension, which is not found in prokaryotic SerRS enzymes. A truncated yeast SES1 gene, lacking the 60 base pairs that encode this C-terminal domain, is able to complement a yeast SES1 null allele strain; thus, the C-terminal extension in SerRS is dispensable for the viability of the cell. However, the removal of the C-terminal peptide affects both stability of the enzyme and its affinity for the substrates. The truncation mutant binds tRNA with 3.6-fold higher affinity, while the Km for serine is 4-fold increased relative to the wild-type SerRS. This indicates the importance of the C-terminal extension in maintaining the overall structure of SerRS.

Substrate selection by aminoacyl-tRNA synthetases.

The integration of genetic and biochemical approaches to study the crystal structure of the glutaminyl-tRNA synthetase (GlnRS):tRNA(Gln):ATP complex has elucidated the mechanism by which GlnRS selects its cognate tRNA for aminoacylation. Three principal types of interaction have been identified: interaction with specific bases in the cognate tRNA, rejection of non-cognate tRNAs, and activation of the active site upon cognate tRNA binding. The recent solving of the crystal structure of tryptophanyl-tRNA synthetase (TrpRS) has allowed comparable studies to be initiated in an aminoacyl-tRNA synthetase which, unlike GlnRS, does not require tRNA binding prior to amino acid activation.

Connecting anticodon recognition with the active site of Escherichia coli glutaminyl-tRNA synthetase.

Escherichia coli glutaminyl-tRNA synthetase (GlnRS) specifically recognizes nucleotides in the anticodon and acceptor stem of tRNA(Gln). Extensive conformational changes in the tRNA(Gln):GlnRS complex and requirement for tRNA in glutaminyl-adenylate formation suggests that accurate anticodon recognition is required for aminoacylation. A 17 amino acid loop in GlnRS (residues 476 to 492) that connects two beta-ribbon motifs was targeted for saturation mutagenesis as the motifs span the anticodon binding domain and extend to the active site. Opal suppressor tRNAs (GLN) derived from tRNA(Gln) are poor substrates for GlnRS, and compensating mutations in glnS (the structural gene for GlnRS) were selected by the ability of the mutant gene product to aminoacylate such a suppressor (GLNA3U70). A number of mutations in loop 476 to 492 were identified by genetic selection, and two of the GlnRS purified mutant enzymes showed elevated specificity constants (kcat/Km) for aminoacylation of a tRNA(Gln)-derived transcript with the opal (UCA) anticodon when compared with the wild-type enzyme. The specificity constants for the mutant enzymes with the cognate tRNA(Gln) transcript (anticodon CUG) were unchanged. Therefore, region 476 to 492 has been identified in communicating anticodon recognition with the active site at a distance of more than 30 A away, supporting a proposed model from the structure of the complex between tRNA(Gln):GlnRS. A previous study has identified residues that interact with the inside of the L-shaped tRNA as communicating accurate anticodon recognition. Therefore, at least two pathways of communication have been identified in the accurate recognition of tRNA by GlnRS.

Thiobacillus ferrooxidans tyrosyl-tRNA synthetase functions in vivo in Escherichia coli.

The tyrosyl-tRNA synthetase gene (tyrZ) from Thiobacillus ferrooxidans, an acidophilic, autotrophic, gram-negative bacterium that participates in bioleaching of minerals, was cloned and sequenced. The encoded polypeptide (TyrRZ) is 407 amino acids in length (molecular mass; 38 kDa). The predicted protein sequence has an extensive overall identity (44%) to the sequence of the protein encoded by the Bacillus subtilus tyrZ gene, one of the two genes encoding tyrosyl-tRNA synthetases in this microorganism. Alignment with Escherichia coli TyrRS revealed limited overall identity (24%), except in the regions of the signature sequence for class I aminoacyl-tRNA synthetases. Complementation of an E. coli strain with a thermosensitive mutation in TyrRS showed that the protein encoded by the T. ferrooxidans tyrZ gene is functional and recognizes the E. coli tRNA(Tyr) as a substrate. TyrZ is a single-copy gene as revealed by Southern blot analysis. The gene was localized upstream from the putative promoters of the rrnT2 ribosomal RNA operon. Although no rho-independent transcription terminator was found between the two genes, a 1.3-kb RNA hybridized to a DNA probe derived from the tyrZ gene. The functional relationship between these two transcription units is discussed.

Coexpression of eukaryotic tRNASer and yeast seryl-tRNA synthetase leads to functional amber suppression in Escherichia coli.

In order to gain insight into the conservation of determinants for tRNA identity between organisms, Schizosaccharomyces pombe and human amber suppressor serine tRNA genes have been examined for functional expression in Escherichia coli. The primary transcripts, which originated from E. coli plasmid promoters, were processed into mature tRNAs, but they were poorly aminoacylated in E. coli and thus were nonfunctional as suppressors in vivo. However, coexpression of cloned Saccharomyces cerevisiae seryl-tRNA synthetase led to efficient suppression in E. coli. This shows that some, but not all, determinants specifying the tRNASer identity are conserved in evolution.

Yeast seryl-tRNA synthetase expressed in Escherichia coli recognizes bacterial serine-specific tRNAs in vivo.

The Saccharomyces cerevisiae serS gene which encodes seryl-tRNA synthetase (SerRS) was expressed in Escherichia coli from the promoter and the ribosome binding sequences contained in its own 5'-flanking region. The low level of yeast SerRS in the prokaryotic host was sufficient to permit in vivo complementation of two temperature-sensitive E. coli serS mutants at the nonpermissive temperature. Thus, yeast SerRS can aminoacylate E. coli tRNA(Ser) species in vivo. Yeast SerRS, isolated from an overexpressing E. coli strain by a rapid two-step purification on FPLC, aminoacylated E. coli tRNA with serine much more poorly (relative kcat/Km = 2 x 10(-4)) than its homologous tRNAs. DL-Serine hydroxamate, an inhibitor of E. coli SerRS, inhibits yeast SerRS in vivo and in vitro with an inhibition constant (Ki) of 2.7 mM, a value 90-fold higher than that for E. coli SerRS.

Acceptor end binding domain interactions ensure correct aminoacylation of transfer RNA.

The recognition of the acceptor stem of tRNA(Gln) is an important element ensuring the accuracy of aminoacylation by Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18). On the basis of known mutations and the crystal structure of the tRNA(Gln).GlnRS complex, we mutagenized at saturation two motifs in the acceptor end binding domain of GlnRS. Mutants with lowered tRNA specificity were then selected in vivo by suppression of a glutamine-specific amber mutation (lacZ1000) with an amber suppressor tRNA derived from tRNA(1Ser). The mischarging GlnRS mutants obtained in this way retain the ability to charge tRNA(Gln), but in addition, they misacylate a number of noncognate amber suppressor tRNAs. The critical residues responsible for specificity are Arg-130 and Glu-131, located in a part of GlnRS that binds the acceptor stem of tRNA(Gln). On the basis of the spectrum of tRNAs capable of being misacylated by such mutants we propose that, in addition to taking part in productive interactions, the acceptor end binding domain contributes to recognition specificity by rejecting noncognate tRNAs through negative interactions. Analysis of the catalytic properties of one of the mischarging enzymes, GlnRS100 (Arg-130-->Pro, Glu-131-->Asp), indicates that, while the kinetic parameters of the mutant enzyme are not dramatically changed, it binds noncognate tRNA(Glu) more stably than the wild-type enzyme does (Kd is 1/8 that of the wild type). Thus, the stability of the noncognate complex may be the basis for mischarging in vivo.

Selectivity and specificity in the recognition of tRNA by E coli glutaminyl-tRNA synthetase.

The specific recognition by Escherichia coli glutaminyl-tRNA synthetase (GlnRS) of tRNA(Gln) is mediated by extensive protein:RNA contacts and changes in the conformation of tRNA(Gln) when complexed with GlnRS. In vivo accuracy of aminoacylation depends on two factors: competition between synthetases, and the context and recognition of identity elements in the tRNA. The structure of the tRNA(Gln):GlnRS complex supports studies from amber and opal suppressor tRNAs, complemented by in vitro aminoacylation of the mutated tRNA transcripts, that the glutamine identity elements are located in the anticodon and acceptor stem of tRNA(Gln). Recognition of individual functional groups in tRNA, for example the 2-amino group of guanosine, is also evident from the result with inosine-substituted tRNAs. Communication between anticodon and acceptor stem recognition is indicated by mutants in GlnRS isolated by genetic selection with opal suppressor tRNAs which are altered in interactions with the inside of the L-shaped tRNA. We have also used genetic selection to obtain mutants of GlnRS altered in acceptor stem recognition with relaxed specificity for amber suppressor tRNAs, and a more extensive mutational analysis shows the importance of the acceptor binding domain to accurate recognition of tRNA.

The recognition of E. coli glutamine tRNA by glutaminyl-tRNA synthetase.

A variety of genetic, biochemical and structural studies have been used to determine factors ensuring the accuracy of recognition by aminoacyl-tRNA synthetases for tRNA. The identity elements of Escherichia coli tRNA(Gln) are located mainly in the anticodon and acceptor stem, and ensure the accurate recognition of the tRNA by glutaminyl-tRNA synthetase. We summarize a number of experimental techniques to define the accuracy of aminoacylation in vivo and in vitro.

Evidence for two types of complexes formed by yeast tyrosyl-tRNA synthetase with cognate and non-cognate tRNA. Effect of ribonucleoside triphosphates.

Polyacrylamide gel electrophoresis at pH 8.3 was used to detect and quantitate the formation of the yeast tyrosyl-tRNA synthetase (an alpha 2-type enzyme) complex with its cognate tRNA. Electrophoretic mobility of the complex is intermediate between the free enzyme and free tRNA; picomolar quantities can be readily detected by silver staining and quantitated by densitometry of autoradiograms when [32P]tRNA is used. Two kinds of complexes of Tyr-tRNA synthetase with yeast tRNA(Tyr) were detected. A slower-moving complex is formed at ratios of tRNA(Tyr)/enzyme less than or equal to 0.5; it is assigned the composition tRNA.(alpha 2)2. At higher ratios, a faster-moving complex is formed, approaching saturation at tRNA(Tyr)/enzyme = 1; any excess of tRNA(Tyr) remains unbound. This complex is assigned the composition tRNA.alpha 2. The slower, i.e. tRNA.(alpha 2)2 complex, but not the faster complex, can be formed even with non-cognate tRNAs. Competition experiments show that the affinity of the enzyme towards tRNA(Tyr) is at least 10-fold higher than that for the non-cognate tRNAs. ATP and GTP affect the electrophoretic mobility of the enzyme and prevent the formation of tRNA.(alpha 2)2 complexes both with cognate and non-cognate tRNAs, while neither tyrosine, as the third substrate of Tyr tRNA synthetase, nor AMP, AMP/PPi, or spermidine, have such effects. Hence, the ATP-mediated formation of the alpha 2 structure parallels the increase in specificity of the enzyme towards its cognate tRNA.

Sequence-specific spin labeling of DNA.

Several DNA fragments deriving from plasmid pBR322 were used to determine the modification sites caused by the reaction with alkylating spin-labeling probes. At a high spin-label concentration, all guanines became alkylated, causing the cleavage of the phosphodiester bonds upon the treatment with piperidine. The lengths of the breakage products of 5'-end labeled DNA treated with spin labels were compared with the length of DNA scission products generated by Maxam-Gilbert procedure for DNA sequence analysis. The distribution of the guanine modifications is dependent on the amount of the reagent used for the alkylation and the ionic conditions of the reaction. The frequency of alkylation by spin labels was greatly enhanced within continuous runs of guanines in DNA. The stabilization of the DNA structure by magnesium or spermine directs the spin-label binding specifically to the most exposed region of DNA fragment containing GGTGG sequence. The sequence-dependent interaction of spin labels with DNA enables the development of the method for the selective spin labeling of DNA molecule.

Deletion analysis in the amino-terminal extension of methionyl-tRNA synthetase from Saccharomyces cerevisiae shows that a small region is important for the activity and stability of the enzyme.

MESI, the structural gene for methionyl-tRNA synthetase from Saccharomyces cerevisiae encodes an amino-terminal extension of 193 amino acids, based on the comparison of the encoded protein with the Escherichia coli methionyl-tRNA synthetase. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA aminoacylation step than on the adenylate formation step. The Km for ATP and methionine are unaltered indicating that the global structure of the enzyme is maintained. The Km for tRNA increased slightly by a factor of 3 which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the Vmax of the enzyme. Examination of the three-dimension structure of the homologous E. coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide-binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagating a mutational alteration to the catalytic residues.