Synthesis of Cysteinyl-tRNA^Cys by a Genome That Lacks the Normal Cysteine-tRNA Synthetase

Abstract

Synthesis of cysteinyl-tRNA(Cys) by cysteine-tRNA synthetase is required for decoding cysteine codons in all known organisms. The genome of the archaeon Methanococcus jannaschii lacks the gene for a normal cysteine-tRNA synthetase. The activity of the enzyme, however, was identified recently, and it allowed the purification of the enzyme and cloning of its gene. Sequence analysis of the gene showed that it encodes proline-tRNA synthetase and, thus, raised the possibility of dual activities in a single aminoacyl-tRNA synthetase. Assays of aminoacyl-adenylate synthesis confirmed the ability of the enzyme to activate proline and cysteine and showed that both activities were independent of tRNA. Assays of tRNA aminoacylation established the specific attachment of proline to tRNA(Pro) and cysteine to tRNA(Cys). However, in contrast to a recent report of comparable activities with cysteine and proline, results here indicate that the adenylate synthesis and aminoacylation activities with cysteine are significantly lower than the respective activity with proline. In addition, there is evidence of overlapping amino acid-binding sites and tRNA-binding sites. These considerations, among others, raised the distinct possibility that the M. jannaschii proline-tRNA synthetase may recruit additional protein or RNA factors to facilitate the synthesis of cysteinyl-tRNA(Cys).

Automated metabolic reconstruction for Methanococcus jannaschii

“…First, it has been shown that M. jannaschii has a dual specificity tRNA synthetase: MJ1238 encodes both prolyl (EC 6.1.1.15) and cysteinyl-tRNA synthetase (EC 6.1.1.16) activities (Bunjun et al 2000, Lipman et al 2000; the cysteinyl-tRNA synthetase activity had not been previously identified (Selkov et al 1997). Second, it has been shown that polyamines are present at high concentrations in M. jannaschii (Graham et al 2000a, Kim et al 2000, Sekowska et al 2000.…”

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

“…This raised the intriguing question of how Cys∼tRNA Cys , an essential component of protein synthesis, is synthesized in these organisms. A number of studies, using incorporation of radiolabeled amino acids into total tRNA, showed that in come cases prolyl-tRNA synthetase (ProRS) from methanogenic archaea could aminoacylate total tRNA with cysteine [46,47], leading to the hypothesis that ProRS attaches proline to tRNA Pro and cysteine to tRNA Cys . In other words, ProRS -hence called ProCysRS -could be responsible for the formation of Cys∼tRNA Cys .…”

How an obscure archaeal gene inspired the discovery of selenocysteine biosynthesis in humans

“…Thus much effort was applied in finding this mysterious CysRS. Two proteins, ProRS (20,21) and MJ1477 (22), were proposed to be the missing CysRS based on in vitro charging experiments and in vivo complementation of an E. coli cysS mutant strain. Later findings placed the complementation data into a different light because the strain retained low CysRS activity (23).…”