Temperature-sensitive mutations in various genes of Escherichia coli K12 can be suppressed by the ssrA gene for 10Sa RNA
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O R I GI N A L P A P E R
H. Nakano á S. Goto á T. Nakayashiki á H. Inokuchi
Temperature-sensitive mutations in various genes of Escherichia coli K12 can be suppressed by the ssrA gene for 10Sa RNA (tmRNA)
Received: 28 September 2000 / Accepted: 5 January 2001 / Published online: 28 February 2001 Ó Springer-Verlag 2001
Abstract An Escherichia coli strain with a deletion in the ssrA gene that encodes 10Sa RNA (tmRNA) was used to screen for temperature-sensitive (ts) mutants whose ts phenotypes were suppressible by introduction of the wild-type ssrA gene. Mutants in four dierent genes were isolated. Ts mutants of this type were also obtained in a screen for mutations in thyA, the structural gene for thymidylate synthase. The ThyA activity in crude extracts prepared from the ts mutants was temperaturesensitive. The presence of the ssrA gene caused an increase in the total amount of the temperature-sensitive enzyme expressed, rather than suppressing the ts activity of the enzyme itself. SsrA-DD, a mutant form of 10Sa RNA, suppressed the ts phenotype of a thyA mutant, suggesting that degradation of a tagged peptide was not required for suppression of the ts phenotype. Considering the fact that ssrA-suppressible mutants could be isolated as temperature-sensitive mutants with mutations in dierent genes, it seems evident that transtranslation can occur on mRNA that is not lacking its stop codon. Keywords ssrA á trans-translation á Temperature-sensitive á thyA
Communicated by H. Ikeda H. Nakano á S. Goto á H. Inokuchi (&) Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan E-mail: [email protected] Tel.: +181-75-753-4200 Fax: +81-75-7534198 T. Nakayashiki Department of Tumor Biology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Introduction 10Sa RNA (also known as tmRNA) is one of the small stable RNAs found in Escherichia coli (Ray and Apirion 1979; Oh and Apirion 1991) and it is encoded by the ssrA gene (Chauhan and Apirion 1989). The 10Sa RNA is 363 bp long and it can form a structure equivalent to a half-molecule of tRNAAla. Actually the 10Sa RNA is aminoacylated with alanine in vitro (Komine et al. 1994; Ushida et al. 1994). The ssrA gene is clearly not essential, because a ssrA deletion strain grows normally at 37°C and 42°C. However the deletion strain does not grow well at 45°C and it exhibits reduced motility on semisolid agar (Komine et al. 1994). Other characteristics of ssrA mutant strains reported include altered growth of certain kimmP22 hybrid phages (Retallack et al. 1994), reduced expression of certain genes (Retallack and Freidman 1995), and increased expression of the Alp protease (Kirby et al. 1994). 10Sa RNA also has a small ORF. It was discovered that a small population of murine interleukin-6 molecules overexpressed in E. coli had an attached peptide tag at the C-terminus, which was identical to the amino sequence encoded by the ORF in 10Sa RNA (Tu et al. 1995). T
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