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

a) tmRNA介导反翻译的分子机制为了研究tmRNA在反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翻译体系。通过体外诱变构建了许多突变体tmrna，并对其tRNA、mRNA和核糖体结合活性进行了测试。从结果中，我们可以确定每种功能所需的tmRNA结构和序列。我们研究了几种抗生素对反翻译的影响：我们发现paromomycin直接与tmRNA结合，导致标签肽翻译的移框。b)反翻译的生理作用我们构建了一个体内系统，可以检测到枯草芽孢杆菌和大肠杆菌的反翻译产物。利用该系统，通过n端氨基酸序列分析分离并鉴定了多个反应产物。用能谱法测定了部分产物的反翻译位点。结果表明，反翻译参与多种细胞活动，如分解代谢抑制、氨基酸代谢、应激反应、产孢等。删除tmRNA基因后，枯草芽孢杆菌的终孢子形成明显受损。遗传和生化实验结果表明，突变体细胞在产孢过程后期的sigma- k合成明显受损。

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.