Effect of glycl-tRNA concentration on in vitro serine incorporation into the peptidoglycan of S. epidermidis

Seven species of seryl-tRNA have been isolated from $\text{S. epidermidis}$ strain Texas 26 by the use of Plaskon CTFE powder as the support for Adogen 464 in reversed-phase chromatography. All but one of these seven seryl-tRNA incorporated ¹⁴C-serine into $\text{in vitro}$ biosynthesized peptidoglycan. This suggested that at least one of the functional roles of six of the seven seryl-tRNA peaks isolated may be involved in $\text{in vivo}$ cell wall synthesis. It has also been shown that the ratio of glycine to serine in the pentapeptide bridge depended upon the concentration of glycyl-tRNA in the assay mixture. By increasing the concentration of glycyl-tRNA the amount of serine inserted into the bridge is increased. Thus the amount of serine incorporated into the pentapeptide bridge appears to be dependent upon the concentration of glycyl-tRNA.     

Transfer of serine into polypeptides and myosin by chromatographic species of seryl-transfer ribonucleic acid.

The efficiencies of two chromatographic species of [3-H]seryl-tRNA, namely peaks I and II, in cell-free amino acid incorporation were investigated. The maximum yield of polypeptide seems to be the same for the reaction mixtures containing either peak I or peak II, suggesting that the efficiency of both peaks in total protein synthesis is the same. The efficiency of transfer of serine into myosin heavy subunit (myosin H) by peaks I and II was also investigated. Peak II of [3-H]seryl-tRNA transfers three times as much serine into myosin H as peak I.     

The characterization of phosphoseryl tRNA from lactating bovine mammary gland.

BD-cellulose and RPC-5 chromatography of tRNA isolated from lactating bovine mammary gland showed the presence of four seryl-tRNA isoacceptors. The species, tRNA IV Ser, with the strongest affinity for BD-cellulose (required ethanol in the elution buffer) could be phosphorylated in the presence of serine, [gamma-32 P]-ATP, seryl-tRNA synthetase and phosphotransferase activity from the same tissue. O-Phosphoserine was identified as the 32P-labelled product after mild alkaline hydrolysis of this aminoacylated tRNA. Pancreatic ribonuclease treatment of the aminoacylated tRNA yielded a labelled product which was identified as phosphoseryladenosine. These results indicated there is a specific phosphoseryl tRNA species in lactating bovine mammary gland. It appears that the formation of phosphoseryl-tRNA proceeds by enzymic phosphorylation of seryl-tRNA.     

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.     

The CUG codon is decoded in vivo as serine and not leucine in Candida albicans.

Previous studies have shown that the yeast Candida albicans encodes a unique seryl-tRNA(CAG) that should decode the leucine codon CUG as serine. However, in vitro translation of several different CUG-containing mRNAs in the presence of this unusual seryl-tRNA(CAG) result in an apparent increase in the molecular weight of the encoded polypeptides as judged by SDS-PAGE even though the molecular weight of serine is lower than that of leucine. A possible explanation for this altered electrophoretic mobility is that the CUG codon is decoded as modified serine in vitro. To elucidate the nature of CUG decoding in vivo, a reporter system based on the C. albicans gene (RBP1) encoding rapamycin-binding protein (RBP), coupled to the promoter of the C. albicans TEF3 gene, was utilized. Sequencing and mass-spectrometry analysis of the recombinant RBP expressed in C. albicans demonstrated that the CUG codon was decoded exclusively as serine while the related CUU codon was translated as leucine. A database search revealed that 32 out of the 65 C. albicans gene sequences available have CUG codons in their open reading frames. The CUG-containing genes do not belong to any particular gene family. Thus the amino acid specified by the CUG codon has been reassigned within the mRNAs of C. albicans. We argue here that this unique genetic code change in cellular mRNAs cannot be explained by the 'Codon Reassignment Theory'.     

Structural and functional investigation of a putative archaeal selenocysteine synthase

Bacterial selenocysteine synthase converts seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec) for selenoprotein biosynthesis. The identity of this enzyme in archaea and eukaryotes is unknown. On the basis of sequence similarity, a conserved open reading frame has been annotated as a selenocysteine synthase gene in archaeal genomes. We have determined the crystal structure of the corresponding protein from Methanococcus jannaschii, MJ0158. The protein was found to be dimeric with a distinctive domain arrangement and an exposed active site, built from residues of the large domain of one protomer alone. The shape of the dimer is reminiscent of a substructure of the decameric Escherichia coli selenocysteine synthase seen in electron microscopic projections. However, biochemical analyses demonstrated that MJ0158 lacked affinity for E. coli seryl-tRNA(Sec) or M. jannaschii seryl-tRNA(Sec), and neither substrate was directly converted to selenocysteinyl-tRNA(Sec) by MJ0158 when supplied with selenophosphate. We then tested a hypothetical M. jannaschii O-phosphoseryl-tRNA(Sec) kinase and demonstrated that the enzyme converts seryl-tRNA(Sec) to O-phosphoseryl-tRNA(Sec) that could constitute an activated intermediate for selenocysteinyl-tRNA(Sec) production. MJ0158 also failed to convert O-phosphoseryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). In contrast, both archaeal and bacterial seryl-tRNA synthetases were able to charge both archaeal and bacterial tRNA(Sec) with serine, and E. coli selenocysteine synthase converted both types of seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). These findings demonstrate that a number of factors from the selenoprotein biosynthesis machineries are cross-reactive between the bacterial and the archaeal systems but that MJ0158 either does not encode a selenocysteine synthase or requires additional factors for activity.     

Identification of a selenocysteyl-tRNA(Ser) in mammalian cells that recognizes the nonsense codon, UGA

The presence of a unique opal suppressor seryl-tRNA in higher vertebrates which is converted to phosphoseryl-tRNA has been known for several years, but its function has been uncertain (see Hatfield, D. (1985) Trends Biochem. Sci. 10, 201-204 for review). In the present study, we demonstrate that this tRNA species also occurs in vivo as selenocysteyl-tRNA(Ser) suggesting that it functions both as a carrier molecule upon which selenocysteine is synthesized and as a direct selenocysteine donor to a growing polypeptide chain in response to specific UGA codons. [75Se]Seleno[3H]cysteyl-tRNA(Ser) formed by administering 75Se and [3H]serine to rat mammary tumor cells (TMT-081-MS) in culture was isolated from the cell extract. The amino acid attached to the tRNA was identified as selenocysteine following its deacylation and reaction with iodoacetate and 3-bromopropionate. The resulting alkyl derivatives co-chromatographed on an amino acid analyzer with authentic carboxymethylselenocysteine and carboxyethylselenocysteine. Seryl-tRNA(Ser) and phosphoseryl-tRNA(Ser) (Hatfield, D., Diamond, A., and Dudock, B. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6215-6219), which co-migrate on a reverse phase chromatographic column with selenocysteyl-tRNA(Ser), were also identified in extracts of TMT-018-MS cells. Hence, we propose that a metabolic pathway for selenocysteine synthesis in mammalian cells is the conversion of seryl-tRNA(Ser) via phosphoseryl-tRNA(Ser) to selenocysteyl-tRNA(Ser). In a ribosomal binding assay selenocysteyl-tRNA(Ser) recognizes UGA but not any of the serine codons. Selenocysteyl-tRNA(Ser) is deacylated more readily than seryl-tRNA(Ser) (i.e. 58% deacylation during 15 min at pH 8.0 and 37 degrees C as compared to 41%).     

Unilateral aminoacylation specificity between bovine mitochondria and eubacteria

The present study shows unilateral aminoacylation specificity between bovine mitochondria and eubacteria (Escherichia coli and Thermus thermophilus) in five amino acid-specific aminoacylation systems. Mitochondrial synthetases were capable of charging eubacterial tRNA as well as mitochondrial tRNA, whereas eubacterial synthetases did not efficiently charge mitochondrial tRNA. Mitochondrial phenylalanyl-, threonyl-, arginyl-, and lysyl-tRNA synthetases were shown to charge and discriminate cognate E. coli tRNA species from noncognate ones strictly, as did the corresponding E. coli synthetases. By contrast, mitochondrial seryl-tRNA synthetase not only charged cognate E. coli serine tRNA species but also extensively misacylated noncognate E. coli tRNA species. These results suggest a certain conservation of tRNA recognition mechanisms between the mitochondrial and E. coli aminoacyl-tRNA synthetases in that anticodon sequences are most likely to be recognized by the former four synthetases, but not sufficiently by the seryl-tRNA synthetase. The unilaterality in aminoacylation may imply that tRNA recognition mechanisms of the mitochondrial synthetases have evolved to be, to some extent, simpler than their eubacterial counterparts in response to simplifications in the species-number and the structural elements of animal mitochondrial tRNAs.     

Peroxin Pex21p interacts with the C-terminal noncatalytic domain of yeast seryl-tRNA synthetase and forms a specific ternary complex with tRNA(Ser)

The seryl-tRNA synthetase from Saccharomyces cerevisiae interacts with the peroxisome biogenesis-related factor Pex21p. Several deletion mutants of seryl-tRNA synthetase were constructed and inspected for their ability to interact with Pex21p in a yeast two-hybrid assay, allowing mapping of the synthetase domain required for complex assembly. Deletion of the 13 C-terminal amino acids abolished Pex21p binding to seryl-tRNA synthetase. The catalytic parameters of purified truncated seryl-tRNA synthetase, determined in the serylation reaction, were found to be almost identical to those of the native enzyme. In vivo loss of interaction with Pex21p was confirmed in vitro by coaffinity purification. These data indicate that the C-terminally appended domain of yeast seryl-tRNA synthetase does not participate in substrate binding, but instead is required for association with Pex21p. We further determined that Pex21p does not directly bind tRNA, and nor does it possess a tRNA-binding motif, but it instead participates in the formation of a specific ternary complex with seryl-tRNA synthetase and tRNA(Ser), strengthening the interaction of seryl-tRNA synthetase with its cognate tRNA(Ser).     

Misacylation of pyrrolysine tRNA in vitro and in vivo

Methanosarcina barkeri inserts pyrrolysine (Pyl) at an in-frame UAG codon in its monomethylamine methyltransferase gene. Pyrrolysyl-tRNA synthetase acylates Pyl onto tRNAPyl, the amber suppressor pyrrolysine Pyl tRNA. Here we show that M. barkeri Fusaro tRNAPyl can be misacylated with serine by the M. barkeri bacterial-type seryl-tRNA synthetase in vitro and in vivo in Escherichia coli. Compared to the M. barkeri Fusaro tRNA, the M. barkeri MS tRNAPyl contains two base changes; a G3:U70 pair, the known identity element for E. coli alanyl-tRNA synthetase (AlaRS). While M. barkeri MS tRNAPyl cannot be alanylated by E. coli AlaRS, mutation of the MS tRNAPyl A4:U69 pair into C4:G69 allows aminoacylation by E. coli AlaRS both in vitro and in vivo.     

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.     

Absence of oligomeric murein intermediates in Escherichia coli.

The intermediates in the biosynthetic pathway of murein were examined in two strains of Escherichia coli to determine whether they synthesized oligomeric precursors in vivo. No oligomeric precursors could be detected; the only intermediates found were the previously described UDP-N-acetylmuramyl peptides, and the two lipid-linked compounds, N-acetylglucosamyl-N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol and N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol. It was concluded that lipid-linked monomers are directly incorporated into the murein sacculus in vivo and do not pass through an oligomeric stage.     

Reinvestigation of phosphorylation of tRNA in Escherichia coli.

This report shows the results of the reinvestigation of tRNA phosphorylation in E. coli. The phosphorylation did not occur on suppressor seryl-tRNA but occurred on other tRNA species. The activity of tRNA phosphorylation was found in E. coli extracts and partially purified. On DEAE-Sephadex A50 and PAGE gel, the phosphorylated-tRNA showed a pattern different from that the natural suppressor serine tRNA.     

The influence of salt concentration on CD spectra of tRNA and aminoacyl-tRNA synthetase

The CD spectra of serine tRNA or seryl-tRNA synthetase were measured. The [theta] values at 210 nm were minimum at 50mM - 0.2M NaCl, at that concentration the velocity of aminoacylation was maximum. This results suggest that A . U and G . C base pairs loosened. The [theta] values at 200 nm decreased according to the decreasing of salt concentration, suggesting the decomposition of A . U base pairs. The CD spectra of seryl-tRNA synthetase at 210-240 nm were not changed in the range of 10mM-0.3M NaCl but the spectra at 260-290 nm showed minimum in the range between 50mM-0.2M NaCl. These results suggest that the influence of salt concentration on the velocity of aminoacylation depends on both the conformational changes of tRNA and seryl-tRNA synthetase.     

The length of the aminoacyl-acceptor stem of the selenocysteine-specific tRNA(Sec) of Escherichia coli is the determinant for binding to elongation factors SELB or Tu

Mutations in selC, which reduce the 8-base pair aminoacyl-acceptor helix to the canonical 7-base pair length (tRNA(Sec)(delAc] or which replace the extra arm of tRNA(Sec) by that of a serine acceptor tRNA species (tRNA(Sec)(ExS), block the function in selenoprotein synthesis in vivo (Baron, C., Heider, J., and B?ck, A. (1990) Nucleic Acids Res. 18, 6761-6766). tRNA(Sec), tRNA(Sec)(delAc), and tRNA(Sec)(ExS) were purified and analyzed for their interaction with purified seryl-tRNA synthetase, selenocysteine synthase and translation factors SELB and EF-Tu. It was found that seryl-tRNA synthetase displays 10-fold impaired Km and Kcat values for tRNA(Sec) in comparison to tRNA(Ser), decreasing the overall charging efficiency (Kcat/Km) of tRNA(Sec) to 1% of that characteristic for tRNA(Ser). tRNA(Sec)(ExS) was a less efficient substrate for the enzyme (Kcat/Km 0.2% of the tRNA(Ser) value) whereas the tRNA(Ser)(delAc) variant was charged with an approximately 2-3-fold improved rate compared to wild-type tRNA(Sec). Both mutant tRNA variants, when charged with L-serine, were able to interact with selenocysteine synthase to give rise to selenocysteyl-tRNA with tRNA(Sec)(ExS) being as efficient as wild-type tRNA(Sec). Seryl-tRNA(Sec)(delAc), on the other hand, was selenylated very slowly. Reduction of the length of the aminoacyl-acceptor stem to 7 base pairs prevented the interaction with translation factor SELB but allowed binding to EF-Tu, irrespective of whether tRNA(Sec)(delAc) was charged with serine or selenocysteine. The aminoacyl-acceptor helix of tRNA(Sec), therefore, is a major determinant directing binding to SELB and precluding interaction with EF-Tu.     

Transfer RNA structural change is a key element in the reassignment of the CUG codon in Candida albicans.

The human pathogenic yeast Candida albicans and a number of other Candida species translate the standard leucine CUG codon as serine. This is the latest addition to an increasing number of alterations to the standard genetic code which invalidate the theory that the code is frozen and universal. The unexpected finding that some organisms evolved alternative genetic codes raises two important questions: how have these alternative codes evolved and what evolutionary advantages could they create to allow for their selection? To address these questions in the context of serine CUG translation in C.albicans, we have searched for unique structural features in seryl-tRNA(CAG), which translates the leucine CUG codon as serine, and attempted to reconstruct the early stages of this genetic code switch in the closely related yeast species Saccharomyces cerevisiae. We show that a purine at position 33 (G33) in the C.albicans Ser-tRNA(CAG) anticodon loop, which replaces a conserved pyrimidine found in all other tRNAs, is a key structural element in the reassignment of the CUG codon from leucine to serine in that it decreases the decoding efficiency of the tRNA, thereby allowing cells to survive low level serine CUG translation. Expression of this tRNA in S.cerevisiae induces the stress response which allows cells to acquire thermotolerance. We argue that acquisition of thermotolerance may represent a positive selection for this genetic code change by allowing yeasts to adapt to sudden changes in environmental conditions and therefore colonize new ecological niches.     

A study of the interaction between tRNASer and seryl-tRNA synthetase from bovine liver

Study by chemical modification of Ser, Arg, His residues and sulfhydryl groups on bovine seryl-tRNA synthetase showed that Ser residues appeared to be unnecessary for the recognition mechanism, but Arg and His residues were essential. It was considered that different sulfhydryl groups related with each recognition of tRNA and ATP. Poly-arginine inhibited the interaction between serine tRNA and SerRS. The CD spectra of a mixture of serine tRNA and poly-arginine indicated that higher-order structure of tRNA changed. Furthermore, the Km and Vmax values of bovine serine isoacceptor, yeast serine tRNA and E. coli serine tRNA for bovine SerRS examined and it was discussed the differences of those base sequences.