In vitro trans-translation of Thermus thermophilus: ribosomal protein S1 is not required for the early stage of trans-translation.
Transfer-messenger RNA (tmRNA) plays a dual role as a tRNA and an mRNA in trans-translation, during which the ribosome replaces mRNA with tmRNA encoding the tag-peptide. These processes have been suggested to involve several tmRNA-binding proteins, including SmpB and ribosomal protein S1. To investigate the molecular mechanism of trans-translation, we developed in vitro systems using purified ribosome, elongation factors, tmRNA and SmpB from Thermus thermophilus. A stalled ribosome in complex with polyphenylalanyl-tRNA(Phe) was prepared as a target of tmRNA. A peptidyl transfer reaction from polyphenylalanyl-tRNA(Phe) to alanyl-tmRNA was observed in an SmpB-dependent manner. The next peptidyl transfer to aminoacyl-tRNA occurred specifically to the putative resume codon for the tag-peptide, which was confirmed by introducing a mutation in the codon. Thus, the in vitro systems developed in this study are useful to investigate the early steps of trans-translation. Using these in vitro systems, we investigated the function of ribosomal protein S1, which has been believed to play a role in trans-translation. Although T. thermophilus S1 tightly bound to tmRNA, as in the case of Escherichia coli S1, it had little or no effect on the early steps of trans-translation.
Takada
Kazuma
K
Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori, Japan.
Takemoto
Chie
C
Kawazoe
Masahito
M
Konno
Takayuki
T
Hanawa-Suetsugu
Kyoko
K
Lee
Sungga
S
Shirouzu
Mikako
M
Yokoyama
Shigeyuki
S
Muto
Akira
A
Himeno
Hyouta
H
D000409:Alanine / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D066298:In Vitro Techniques; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D016963:Thermus thermophilus / Q000378:metabolism*
D003062:Codon; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C015494:ribosomal protein S1; D010649:Phenylalanine; D000409:Alanine