In vitro trans-translation of Thermus thermophilus: Ribosomal protein S1 is not required for the early stage of trans-translation

• DOI: 10.1261/rna.363207
• Publication Date: 2007-02-14T02:58:22Z
• Authors: Kazuma Takada; Chie Takemoto; Masahito Kawazoe; Takayuki Konno; Kyoko Hanawa-Suet...; SungGa Lee; Mikako Shirouzu; Shigeyuki Yokoyama; Akira Muto; Hyouta Himeno
• Keywords: Ribosomal Proteins; 0303 health sciences; Alanine; Binding Sites; Phenylalanine; Thermus thermophilus; RNA-Binding Proteins; In Vitro Techniques; RNA, Transfer, Amino Acyl; RNA, Bacterial; 03 medical and health sciences; Protein Biosynthesis; Mutation; Escherichia coli; Codon; Protein Binding

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-tRNAPhe was prepared as a target of tmRNA. A peptidyl transfer reaction from polyphenylalanyl-tRNAPhe 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.