1: J Bacteriol. 2004 Jan;186(1):8-14.

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

Ruan B, Nakano H, Tanaka M, Mills JA, DeVito JA, Min B, Low KB, Battista JR, Soll
D.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, 
Connecticut 06520-8114, USA.

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. Soll, 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 Crecy-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.

PMCID: PMC303452
PMID: 14679218 [PubMed - indexed for MEDLINE]

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