Abstract
Methanocaldococcus jannaschii prolyl-tRNA synthetase (ProRS) was previously reported to also catalyze the synthesis of cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) to make up for the absence of the canonical cysteinyl-tRNA synthetase in this organism (Stathopoulos, C., Li, T., Longman, R., Vothknecht, U. C., Becker, H., Ibba, M., and Söll, D. (2000) Science 287, 479-482; Lipman, R. S., Sowers, K. R., and Hou, Y. M. (2000) Biochemistry 39, 7792-7798). Here we show by acid urea gel electrophoresis that pure heterologously expressed recombinant M. jannaschii ProRS misaminoacylates M. jannaschii tRNA(Pro) with cysteine. The enzyme is unable to aminoacylate purified mature M. jannaschii tRNA(Cys) with cysteine in contrast to facile aminoacylation of the same tRNA with cysteine by Methanococcus maripaludis cysteinyl-tRNA synthetase. Although M. jannaschii ProRS catalyzes the synthesis of Cys-tRNA(Pro) readily, the enzyme is unable to edit this misaminoacylated tRNA. We discuss the implications of these results on the in vivo activity of the M. jannaschii ProRS and on the nature of the enzyme involved in the synthesis of Cys-tRNA(Cys) in M. jannaschii.
Highlights
Cysteinyl-tRNACys (Cys-tRNACys)1 is an essential component of the translational machinery of all organisms for the incorporation of cysteine into proteins
We discuss the implications of these results on the in vivo activity of the M. jannaschii prolyl-tRNA synthetase (ProRS) and on the nature of the enzyme involved in the synthesis of Cys-tRNACys in M. jannaschii
M. jannaschii ProRS Aminoacylates tRNAPro with Cysteine— Acid urea gel electrophoresis has been a reliable tool to determine the aminoacylation specificity of aminoacyl-tRNA synthetases [19]
Summary
Cys-tRNACys, cysteinyl-tRNACys; CysRS, cysteinyl-tRNA synthetase; ProRS, prolyl-tRNA synthetase; NiNTA, nickel-nitrilotriacetic acid; ProRS⌬50, carboxyl-terminal deletion of M. jannaschii ProRS. The reports that purified recombinant M. jannaschii prolyl-tRNA synthetase (ProRS) was able to aminoacylate tRNACys with cysteine provided an apparent answer to this question (6 –11). This led to the notion of ProRS as an unusual dual specificity enzyme capable of attaching proline to tRNAPro and cysteine to tRNACys [6, 7]; the enzyme was named ProCysRS [12]. We show that a purified recombinant form of the M. jannaschii enzyme, previously called ProCysRS, misaminoacylates tRNAPro with cysteine but is unable to aminoacylate tRNACys with cysteine These properties of M. jannaschii ProRS raise the question of the true in vivo activities of this enzyme. In view of these results, we have renamed the enzyme ProRS in this paper
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