Cysteinyl-tRNA(Cys) formation in Methanocaldococcus jannaschii: the mechanism is still unknown - PubMed (original) (raw)

Cysteinyl-tRNA(Cys) formation in Methanocaldococcus jannaschii: the mechanism is still unknown

Benfang Ruan et al. J Bacteriol. 2004 Jan.

Abstract

Most organisms form Cys-tRNA(Cys), an essential component for protein synthesis, through the action of cysteinyl-tRNA synthetase (CysRS). However, the genomes of Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, and Methanopyrus kandleri do not contain a recognizable cysS gene encoding CysRS. It was reported that M. jannaschii prolyl-tRNA synthetase (C. Stathopoulos, T. Li, R. Longman, U. C. Vothknecht, H. D. Becker, M. Ibba, and D. Söll, Science 287:479-482, 2000; R. S. Lipman, K. R. Sowers, and Y. M. Hou, Biochemistry 39:7792-7798, 2000) or the M. jannaschii MJ1477 protein (C. Fabrega, M. A. Farrow, B. Mukhopadhyay, V. de Crécy-Lagard, A. R. Ortiz, and P. Schimmel, Nature 411:110-114, 2001) provides the "missing" CysRS activity for in vivo Cys-tRNA(Cys) formation. These conclusions were supported by complementation of temperature-sensitive Escherichia coli cysS(Ts) strain UQ818 with archaeal proS genes (encoding prolyl-tRNA synthetase) or with the Deinococcus radiodurans DR0705 gene, the ortholog of the MJ1477 gene. Here we show that E. coli UQ818 harbors a mutation (V27E) in CysRS; the largest differences compared to the wild-type enzyme are a fourfold increase in the K(m) for cysteine and a ninefold reduction in the k(cat) for ATP. While transformants of E. coli UQ818 with archaeal and bacterial cysS genes grew at a nonpermissive temperature, growth was also supported by elevated intracellular cysteine levels, e.g., by transformation with an E. coli cysE allele (encoding serine acetyltransferase) or by the addition of cysteine to the culture medium. An E. coli cysS deletion strain permitted a stringent complementation test; growth could be supported only by archaeal or bacterial cysS genes and not by archaeal proS genes or the D. radiodurans DR0705 gene. Construction of a D. radiodurans DR0705 deletion strain showed this gene to be dispensable. However, attempts to delete D. radiodurans cysS failed, suggesting that this is an essential Deinococcus gene. These results imply that it is not established that proS or MJ1477 gene products catalyze Cys-tRNA(Cys) synthesis in M. jannaschii. Thus, the mechanism of Cys-tRNA(Cys) formation in M. jannaschii still remains to be discovered.

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Figures

FIG. 1.

FIG. 1.

Scheme of cassette constructs for Deinococcus gene deletions. (A) DR0705 in the D. radiodurans R1 chromosome. (B) DR0705 deletion cassette. (C) DR1670 deletion cassette. Primers indicated above or below the cassette were used for PCR. Sections of the diagrams are labeled as follows: A upstream, 971 bp immediately upstream of the initiation codon of DR0705; A downstream, 806 bp immediately downstream of the termination codon of DR0705; B upstream, 886 bp immediately upstream of the initiation codon of DR1670; B downstream, 957 bp immediately downstream of the termination codon of DR1670.

FIG. 2.

FIG. 2.

Growth of E. coli cysS(Ts) strain UQ818 at 41°C on a cysteine gradient λ plate. The highest cysteine concentration is in the well (see Materials and Methods).

FIG. 3.

FIG. 3.

Complementation of E. coli cysS(Ts) strain UQ818 with pCYB1, pCYB-EC_cysS_, pCYB-cysE_M256I, or pCYB-MM_cysS. LB agar containing ampicillin and IPTG was used for growth at 41°C (see Materials and Methods).

FIG. 4.

FIG. 4.

Replacement of the D. radiodurans DR0705 gene with the P_katA-npt_ cassette. Lanes 1 and 10, DNA size markers; lanes 2, 4, 6, and 8, DNA from D. radiodurans R1; lanes 3, 5, 7, and 9, DNA from DR0705 deletion strain TNK201. Lanes: 2 and 3, PCR products of primers AFW4 and ARV4; 4 and 5, PCR products of primers AFW2 and ARV3; 6 and 7, _Hin_dIII digestion of DNA in lanes 4 and 5, respectively; 8 and 9, _Xho_I digestion of DNA in lanes 4 and 5, respectively. DR0705 has no restriction sites for _Hin_dIII and _Xho_I, while the npt gene is cut once by both enzymes.

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