Molecular mechanism of trans-translation mediated by tmRNA and its application

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a) Molecular mechanism of trans-translation mediated by tmRNA To investigate the functional structure of tmRNA in trans-translation reaction, we have developed an in vitro trans-translation system using E. coli extracts. Many mutant tmRNAs were constructed by in vitro mutagenesis, and tested their activities as tRNA, mRNA and rribosome binding. From the results, we could identify the structures and sequences in tmRNA required for each function. The effect of several antibiotics on the trans-translation was investigated: we found that paromomycin directly binds to tmRNA causing the frame-shift of translation of tag-peptides.b) Physiological roles of trans-translation We have constructed an in vivo system in which trans-translation products can be detected in Bacillus subtilis and E. coli. Using this system, several reaction products were isolated and identified by the N-terminal amino acid sequence analyses. The sites of trans-translation in some of the products were determined by MAS-spectrometry. It was shown that the trans-translation is involved in various cellular activities, such as catabolite repression, amino acid metabolism, stress responses, sporulation and so on.Endspore formation of Bacillus subtilis strain is significantly impaired by deleting the gene for tmRNA. The genetic and biochemical experimental results revealed that the synthesis of sigma-Kduring the late stage of sporulation process is significantly impaired in the mutant cells.