Article

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

Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori, Japan.
RNA (impact factor: 5.09). 05/2007; 13(4):503-10. DOI:10.1261/rna.363207 pp.503-10
Source: PubMed

ABSTRACT 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.

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Keywords

alanyl-tmRNA
 
dual role
 
elongation factors
 
Escherichia coli S1
 
next peptidyl transfer
 
peptidyl transfer reaction
 
polyphenylalanyl-tRNA(Phe)
 
processes
 
purified ribosome
 
putative
 
ribosomal protein S1
 
SmpB
 
SmpB-dependent manner
 
stalled ribosome
 
T. thermophilus S1
 
Thermus thermophilus
 
tmRNA encoding
 
tmRNA-binding proteins
 
Transfer-messenger RNA
 
vitro systems