© 2005 The Japanese Biochemical Society.
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Interaction Analysis between tmRNA and SmpB from Thermus thermophilus
1 Department of Industrial Chemistry, Faculty of Engineering, Chiba Institute of Technology, Chiba 275-0016; 2 RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045; 3 Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904; 4 Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561; 5 RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki, Sayo, Hyogo 679-5148; 6 Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043; and 7 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033
To whom correspondence should be addressed. Tel: +81-474-78-0425, Fax: +81-474-78-0425, E-mail: gkawai{at}sea.it-chiba.ac.jp
Small protein B, SmpB, is a tmRNA-specific binding protein essential for trans-translation. We examined the interaction between SmpB and tmRNA from Thermus thermophilus, using biochemical and NMR methods. Chemical footprinting analyses using full-length tmRNA demonstrated that the sites protected upon SmpB binding are located exclusively in the tRNA-like domain (TLD) of tmRNA. To clarify the SmpB binding sites, we constructed several segments derived from TLD. Optical biosensor interaction analyses and melting profile analyses with mutational studies showed that SmpB efficiently binds to only a 30-nt segment that forms a stem and loop, with the 5' and 3' extensions composed of the D-loop and variable-loop analogues. The conserved sequences, 16UCGA and 319GAC, in the extensions are responsible for the SmpB binding. These results agree with the those visualized by the cocrystal structure of TLD and SmpB from Aquifex aeolicus. In addition, NMR chemical shift mapping analyses, using the 30-nt segment and 15N-labeled SmpB, revealed the characteristic RNA binding mode. The hydrogen bond pattern around ß2 changes, with the Gly in ß2, which acts as a hinge, showing the largest chemical shift change. It appears that SmpB undergoes structural changes indicating an induced fit upon binding to the specific region of TLD.
* Present address: Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu-shi, Gunma 376-8515.
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