In vitro trans-translation of Thermus thermophilus: Ribosomal protein S1 is not required for the early stage of trans-translation
- Kazuma Takada1,2,
- Chie Takemoto3,
- Masahito Kawazoe3,
- Takayuki Konno1,2,
- Kyoko Hanawa-Suetsugu2,3,
- SungGa Lee1,6,
- Mikako Shirouzu3,
- Shigeyuki Yokoyama3,4,
- Akira Muto1,2,5, and
- Hyouta Himeno1,2,5
- 1Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori 036-8561, Japan
- 2The United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8551, Japan
- 3Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan
- 4Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 133-0033, Japan
- 5RNA Research Center, Hirosaki University, Hirosaki, Aomori 036-8561, Japan
Abstract
Transfer-messenger RNA (tmRNA) plays a dual role as a tRNA and an mRNA in trans-translation, during which the ribosome replaces mRNA with tmRNA encoding the tag-peptide. These processes have been suggested to involve several tmRNA-binding proteins, including SmpB and ribosomal protein S1. To investigate the molecular mechanism of trans-translation, we developed in vitro systems using purified ribosome, elongation factors, tmRNA and SmpB from Thermus thermophilus. A stalled ribosome in complex with polyphenylalanyl-tRNAPhe was prepared as a target of tmRNA. A peptidyl transfer reaction from polyphenylalanyl-tRNAPhe to alanyl-tmRNA was observed in an SmpB-dependent manner. The next peptidyl transfer to aminoacyl-tRNA occurred specifically to the putative resume codon for the tag-peptide, which was confirmed by introducing a mutation in the codon. Thus, the in vitro systems developed in this study are useful to investigate the early steps of trans-translation. Using these in vitro systems, we investigated the function of ribosomal protein S1, which has been believed to play a role in trans-translation. Although T. thermophilus S1 tightly bound to tmRNA, as in the case of Escherichia coli S1, it had little or no effect on the early steps of trans-translation.
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Footnotes
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↵6 Present address: Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
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Reprint requests to: Hyouta Himeno; Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori 036-8561, Japan; e-mail: himeno{at}cc.hirosaki-u.ac.jp; fax: 81-172-39-3593.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.363207.
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- Received October 27, 2006.
- Accepted January 2, 2007.
- Copyright © 2007 RNA Society