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

A)tmRNA介导的反式翻译的分子机制为了研究tmRNA在反式翻译反应中的功能结构，我们利用大肠杆菌提取物建立了一个体外反式翻译系统。通过体外诱变构建了许多突变型tmRNAs，并检测了它们的tRNA、mRNA和核糖体结合活性。从结果中，我们可以确定tmRNA中每种功能所需的结构和序列。研究了几种抗生素对反式翻译的影响：我们发现帕罗霉素直接与tmRNA结合，导致标签肽翻译的移码。b)反式翻译的生理作用我们构建了一个体内系统，可以在枯草芽孢杆菌和大肠杆菌中检测到反式翻译产物。利用该体系对几种反应产物进行了N-端氨基酸序列分析，并对其进行了鉴定。用MAS-光谱分析确定了部分产物中的反式翻译位点。研究表明，该基因参与了多种细胞活动，如分解代谢抑制、氨基酸代谢、应激反应、产孢量等。缺失该基因可显著抑制枯草芽孢杆菌的终孢子形成。遗传和生化实验结果表明，突变细胞在产孢子过程的后期合成Sigma-K的能力明显受损。

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.