title: Single molecule imaging of the trans-translation entry process via anchoring of the tagged ribosome.
abstract: 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.
authors.affiliation: Graduate School of Frontier Science, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8562, Japan.
authors.forename: Zhan-Ping
authors.initials: ZP
authors.lastname: Zhou
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Shimizu
authors.forename: Hisashi
authors.initials: H
authors.lastname: Tadakuma
authors.forename: Hideki
authors.initials: H
authors.lastname: Taguchi
authors.forename: Koichi
authors.initials: K
authors.lastname: Ito
authors.forename: Takuya
authors.initials: T
authors.lastname: Ueda
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D057054:Molecular Imaging / Q000379:methods; D009690:Nucleic Acid Conformation; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008024:Ligands; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C030004:ribosomal protein S2; C040469:ribosomal protein S30; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu