分枝杆菌中反义RNA靶向tmRNA介导的反式翻译调控新机制

蛋白质合成时，很多因素导致核糖体无法及时与mRNA分离而停滞。为此，细菌进化出核糖体拯救系统，对停滞在mRNA上的核糖体进行回收利用。以tmRNA为核心的反式翻译是细菌中主要的核糖体拯救系统。tmRNA类似携带丙氨酰的tRNA，识别受困的核糖体并致其移位，核糖体继续以tmRNA的ORF为模板在缺陷肽C端翻译出降解标签肽，以完成核糖体拯救并促进缺陷肽水解。但目前尚无tmRNA调控机制研究的报道。申请人通过转录组分析，在多种细菌中发现了tmRNA的反义RNA，命名为astmRNA。本项目拟以耻垢分枝杆菌为出发菌株，研究细胞内外信号及转录因子对astmRNA的调控，揭示其受到上游调控后，进一步通过调节tmRNA的转录量、稳定性以影响其生理功能的作用机制。本项目首次发现astmRNA，并对tmRNA的调控机制进行深入研究，不仅深刻阐明了反式翻译拯救系统的调控机制，而且为药物靶标的设计提供新思路。

Bacteria devote a large percentage of their resources to protein synthesis, which therefore must be protected delicately. For example, ribosomes that stall during translation need to be rescued to ensure that the protein synthesis capacity of the cell is maintained. Stalling arises when ribosomes become trapped at the 3’-end of an mRNA, which occurs when a codon is unsuitable, leading to the synthesis arrest during protein elongation or termination. Almost all bacteria use a mechanism known as trans-translation, which is mediated by tmRNA, to rescue stalled ribosomes. During trans-translation, alanine charged tmRNA recognizes the arrested ribosome to drive its translocation. The ribosome then resumes protein translation by utilizing an open reading frame (ORF) within tmRNA to provide a stop codon for translation termination and rescue of trapped ribosomes. In this manner, the nascent polypeptide is attached with a proteolysis tag, and the chimeric polypeptide is released from the ribosome, followed by degradation with specific ATP dependent proteases. Despite its well established importance, the regulation of tmRNA has rarely been investigated. In a recent study, by analyzing the transcriptome data, we found the existence of a cis-acting antisense RNA (hereafter referred to as “astmRNA”) encoded via the opposite strand of tmRNA in many bacteria. We have verified the transcription of astmRNA in M. smegmatis by specific primer-mediated reverse PCR, promoter activity, and RACE assays. In this project, we devote to study how does astmRNA regulates the expression, stability, and biological function of tmRNA in response to various intra/extracellular cues. We also aim to identify the upstream transcription regulators of astmRNA to examine the influence of tmRNA mediated tran-translation when astmRNA is being regulated. Besides, this project will also explore the biological significance of astmRNA and analyze its distribution and evolution among bacteria. We are the first group to identify the astmRNA and will further conduct in-depth research on the regulation mechanism of tmRNA. It will not only profoundly clarify the regulation mechanism of trans-translation rescue system but also provide ideas for the design of new drug targets and development of new vaccines.