Trypanosoma seryl-tRNA synthetase is a metazoan-like enzyme with high affinity for tRNASec

Trypanosomatids are important human pathogens that form a basal branch of eukaryotes. Their evolutionary history is still unclear as are many aspects of their molecular biology. Here we characterize essential components required for the incorporation of serine and selenocysteine into the proteome of Trypanosoma. First, the biological function of a putative Trypanosoma seryl-tRNA synthetase was characterized in vivo. Secondly, the molecular recognition by Trypanosoma seryl-tRNA synthetase of its cognate tRNAs was dissected in vitro. The cellular distribution of tRNA(Sec) was studied, and the catalytic constants of its aminoacylation were determined. These were found to be markedly different from those reported in other organisms, indicating that this reaction is particularly efficient in trypanosomatids. Our functional data were analyzed in the context of a new phylogenetic analysis of eukaryotic seryl-tRNA synthetases that includes Trypanosoma and Leishmania sequences. Our results show that trypanosomatid seryl-tRNA synthetases are functionally and evolutionarily more closely related to their metazoan homologous enzymes than to other eukaryotic enzymes. This conclusion is supported by sequence synapomorphies that clearly connect metazoan and trypanosomatid seryl-tRNA synthetases.     

tRNA-dependent amino acid discrimination by yeast seryl-tRNA synthetase.

The ability of aminoacyl-tRNA synthetases to distinguish between similar amino acids is crucial for accurate translation of the genetic code. Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) employs tRNA-dependent recognition of its cognate amino acid serine [Lenhard, B., Filipic, S., Landeka, I., Skrtic, I., S?ll, D. & Weygand-Durasevic, I. (1997) J. Biol. Chem.272, 1136-1141]. Here we show that dimeric SerRS enzyme complexed with one molecule of tRNASer is more specific and more efficient in catalyzing seryl-adenylate formation than the apoenzyme alone. Sequence-specific tRNA-protein interactions enhance discrimination of the amino acid substrate by yeast SerRS and diminish the misactivation of the structurally similar noncognate threonine. This may proceed via a tRNA-induced conformational change in the enzyme's active site. The 3'-terminal adenosine of tRNASer is not important in effecting the rearrangement of the serine binding site. Our results do not provide an indication for a readjustment of ATP binding in a tRNA-assisted manner. The stoichiometric analyses of the complexes between the enzyme and tRNASer revealed that two cognate tRNA molecules can be bound to dimeric SerRS, however, with very different affinities.     

Cloning and characterization of the gene for Escherichia coli seryl-tRNA synthetase.

Seryl-tRNA synthetase is the gene product of the serS locus in Escherichia coli. Its gene has been cloned by complementation of a serS temperature sensitive mutant K28 with an E. coli gene bank DNA. The resulting clones overexpress seryl-tRNA synthetase by a factor greater than 50 and more than 6% of the total cellular protein corresponds to the enzyme. The DNA sequence of the complete coding region and the 5'- and 3' untranslated regions was determined. Protein sequence comparison of SerRS with all available aminoacyl-tRNA synthetase sequences revealed some regions of significant homology particularly with the isoleucyl- and phenylalanyl-tRNA synthetases from E. coli.     

Characterization of a temperature-sensitive Escherichia coli mutant and revertants with altered seryl-tRNA synthetase activity.

A mutation in the structural gene coding for seryl-tRNA synthetase in temperature-sensitive Escherichia coli K28 has been reported to alter the level of enzyme expression at high temperature (R. J. Hill and W. Konigsberg, J. Bacteriol. 141:1163-1169, 1980). We identified this mutation as a C-->T transition in the first base of codon 386, resulting in a replacement of histidine by tyrosine. The steady-state levels of serS mRNA in K28 and in the wild-type strains are very similar. Pulse-chase labeling experiments show a difference in protein stability, but not one important enough to account for the temperature sensitivity of K28. The main reason for the temperature sensitivity of K28 appears to be the low level of specific activity of the mutant synthetase at nonpermissive temperature, not a decreased expression level. Spontaneous temperature-resistant revertants were selected which were found to have about a fivefold-higher level of SerRS than the K28 strain. We identified the mutation responsible for the reversion as being upstream from the -10 sequence in the promoter region. The steady-state levels of serS mRNA in the revertants are significantly higher than that in the parental strain.     

Isolation and characterization of an Escherichia coli seryl-tRNA synthetase mutant with a large increase in Km for serine.

A mutant of Escherichia coli resistant to serine hydroxamate which has a large increase in Km for serine of seryl-tRNA synthetase is described. The mutant serS gene was cloned and sequenced and was found to contain a single-base-pair mutation, resulting in the substitution of the residue alanine 262 by valine in motif 2. The methyl side chain of alanine 262 is not exposed at the active site, and molecular modeling indicated that replacement of alanine 262 by valine does not significantly affect the configuration of amino acids at the active site. This finding suggests that the residue at this position may be involved in a conformational change (possibly induced by ATP binding) which is necessary for optimal binding of the cognate amino acid.     

Crystals of seryl-tRNA synthetase from Escherichia coli. Preliminary crystallographic data

Crystals of seryl-tRNA synthetase from Escherichia coli can be grown from ammonium sulphate/octyl glucoside solutions in two days. The crystals appear to be very suitable for X-ray analysis, diffracting to at least 2.8 A resolution and being resistant to radiation damage. The crystals are monoclinic (space group C2) with cell parameters a = 148.2 A, b = 90.6 A, c = 69.5 A and beta = 119.0 degrees. Depending on whether the asymmetric unit is the enzyme monomer (Mr 48,414) or dimer the Vm value would be either 4.12 or 2.10 A3/dalton. Although the former would indicate a rather high solvent content, other proteins crystallized in the presence of octyl glucoside have Vm values similar to this.     

Crystals of seryl-tRNA synthetase from Thermus thermophilus. Preliminary crystallographic data

Crystals have been obtained of seryl-tRNA synthetase from the extreme thermophile Thermus thermophilus, using mixed solutions of ammonium sulphate and methane pentane diol. The crystals are very stable and diffract to at least 2 A. The crystals are monoclinic (space group P21) with cell parameters a = 87.1 A, b = 126.9 A, c = 63.5 A and beta = 109.7 degrees.     

The proximal region of a noncatalytic eukaryotic seryl-tRNA synthetase extension is required for protein stability in vitro and in vivo

Eukaryotic cytosolic seryl-tRNA synthetases (SerRS) have idiosyncratic C-terminal extensions not present in prokaryotic counterparts. The extensions of two eukaryotic SerRSs were subjected to mutagenesis and partial truncation. Only minor parts of the yeast or maize SerRS extensions, adjacent to the catalytic core (7 of 20 and 8 of 26 amino acids, respectively), were found to be indispensable for protein stability. Truncated proteins with substantially shortened extensions displayed unaltered catalytic properties and could complement a Saccharomyces cerevisiae strain with a disrupted SerRS gene, if these proximal regions were left intact. Although the yeast C-terminal SerRS extension is required for Pex21p binding, the maize counterpart with an appended yeast SerRS extension remained incapable of Pex21p binding, implying that additional regions of yeast SerRS may also contribute to the interaction with the peroxin. The proximal region of the eukaryotic SerRS C-terminal extension is indispensable for protein stability, while the remaining part of the extension remains available for other functions, such as species-specific protein:protein interactions.     

Superposition of a tRNASer acceptor stem microhelix into the seryl-tRNA synthetase complex

Aminoacyl-tRNA synthetases catalyze the formation of aminoacyl-tRNAs. Seryl-tRNA synthetase is a class II synthetase, which depends on rather few and simple identity elements in tRNA(Ser) to determine the amino acid specificity. tRNA(Ser) acceptor stem microhelices can be aminoacylated with serine, which makes this part of the tRNA a valuable tool for investigating the structural motifs in a tRNA(Ser)-seryl-tRNA synthetase complex. A 1.8A-resolution tRNA(Ser) acceptor stem crystal structure was superimposed to a 2.9A-resolution crystal structure of a tRNA(Ser)-seryl-tRNA synthetase complex for a visualization of the binding environment of the tRNA(Ser) microhelix.