Contribution of the second OB fold of ribosomal protein S1 from Escherichia coli to the recognition of TmRNA.
Escherichia coli ribosomal protein S1 is composed of six repeating homologous oligonucleotide/oligosaccharide-binding fold (OB folds). In trans-translation, S1 plays a role in delivering transfer-messenger RNA (tmRNA) to stalled ribosomes. The second OB fold of S1 was found to be protected from tryptic digestion in the presence of tmRNA. Truncated S1 mutant Delta2, in which the first and second OB folds were deleted, showed significantly decreased tmRNA-binding activity. Furthermore, the E. coli S1 homolog (BS1) from Bacillus subtilis, which corresponds to the four C-terminal OB folds of E. coli S1, showed no interaction with E. coli tmRNA, as judged by the results of a gel shift assay. Surface plasmon resonance analysis revealed that mutant Delta2 and BS1 had decreased association rate constants (ka, 0.59 x 10(3) M(-1).S(-1); and ka, 1.89 x 10(3) M(-1).S(-1)), while they retained the respective dissociation rate constants (kd, 0.67 x 10(-3) S(-1); and kd, 0.53 x 10(-3) S(-1)), in comparison with wild-type protein S1 (ka, 3.32 x 10(3) M(-1).S(-1); and kd, 0.56 x 10(-3) S(-1)). These results suggest that the second OB fold in protein S1 is essential for the recognition of tmRNA, while the four C-terminal OB folds play a role in stabilizing the S1-tmRNA complex.
Okada
Takahiro
T
Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Wower
Iwona K
IK
Wower
Jacek
J
Zwieb
Christian W
CW
Kimura
Makoto
M
D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D017353:Gene Deletion; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation / Q000235:genetics / Q000502:physiology; D010957:Plasmids; D011487:Protein Conformation; D017510:Protein Folding; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics; D020349:Surface Plasmon Resonance; D014357:Trypsin
D012329:RNA, Bacterial; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C015494:ribosomal protein S1; D012343:RNA, Transfer; D014357:Trypsin