A functional interaction of SmpB with tmRNA for determination of the resuming point of trans-translation
- Takayuki Konno1,2,3,
- Daisuke Kurita1,2,
- Kazuma Takada1,2,
- Akira Muto1,2,4, and
- Hyouta Himeno1,2,4
- 1Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan
- 2The United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8550, Japan
- 3Department of Microbiology, AKITA Prefectural Research Center for Public Health and Environment, Akita 010-0874, Japan
- 4RNA Research Center, Hirosaki University, Hirosaki 036-8561, Japan
Abstract
In trans-translation, transfer-messenger RNA (tmRNA), possessing a dual function as a tRNA and an mRNA, relieves a stalled translation on the ribosome with the help of SmpB. Here, we established an in vitro system using Escherichia coli translation and trans-translation factors to evaluate two steps of trans-translation, peptidyl transfer from peptidyl-tRNA to alanyl-tmRNA and translation of the resume codon on tmRNA. Using this system, the effects of several mutations upstream of the tag-encoding region on tmRNA were examined. These mutations affected translation of the resume codon rather than peptidyl transfer, and one of them, A84U/U85G, caused a shift of the resume codon by −1. We also found that U85 is protected from chemical modification by SmpB. In the A84U/U85G mutant, the base of protection was shifted from 85 to 84. Another mutation, A86U, which caused a shift of the resume codon by +1, shifted the base of protection from 85 to 86. The protection at 85 was suppressed by a mutation in the tRNA-like domain critical to SmpB binding. These results suggest that SmpB serves to bridge two separate domains of tmRNA to determine the initial codon for tag-translation. A mutant SmpB with a truncation of the unstructured C-terminal tail failed to promote peptidyl transfer, although it still protected U85 from chemical modification.
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Footnotes
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Reprints requests to: Hyouta Hiemno, Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 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.604907.
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- Received April 23, 2007.
- Accepted July 5, 2007.
- Copyright © 2007 RNA Society