Single molecule imaging of the trans-translation entry process via anchoring of the tagged ribosome

Zhan Ping Zhou*, Yoshihiro Shimizu, Hisashi Tadakuma, Hideki Taguchi, Koichi Ito, Takuya Ueda

*この研究の対応する著者

研究成果: Article査読

18 被引用数 (Scopus)

抄録

Trans-translation is an eubacterial quality control system to rescue the stalled ribosome, in which multiple components such as transfer messenger RNA (tmRNA) and Small protein B (SmpB) are involved. However, how these molecules interact with ribosome remains elusive. Here, we report the single molecule analysis of the trans-translation process. We developed a new method to label the functional ribosome, in which a tag protein (the HaloTag protein of 297 amino acids) was fused to the 30S ribosomal protein S2 and covalently labelled with specific ligand (HaloTag ligand), resulting in the stable and specific labelling of ribosome. Ribosomes were anchored onto the glass surface using biotinylated derivative of the Cy3 HaloTag ligand (i.e. biotin-Cy3-ligand), and real-time interactions of Cy5-tmRNA/SmpB/EF-Tu ternary complexes with anchored ribosomes are observed as a model of the trans-translation entry. Statistical analysis revealed that Cy5-tmRNA/SmpB/EF-Tu ternary complexes bind to the anchored ribosome with the second-order rate constant of 2.6 × 10 6 (1/M/s) and tmRNAs undergo multi-modal pathway before release from ribosome. The methods presented here are also applicable to the analysis for general translation processes. The Authors 2011. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved2011

本文言語English
ページ(範囲)609-618
ページ数10
ジャーナルJournal of biochemistry
149
5
DOI
出版ステータスPublished - 2011 5
外部発表はい

ASJC Scopus subject areas

  • 生化学
  • 分子生物学

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