Transfer-messenger RNA (tmRNA) is ubiquitous in bacteria, acting as the core component for the trans-translation system that contributes to label the aberrantly synthesized peptides for degradation and to release the stalled ribosomes. Deletion of tmRNA causes a variety of phenotypes related to important physiological processes in bacteria. To illustrate the molecular mechanism of the versatility of tmRNA in aquatic pathogen Aeromonas veronii, we mutated the C-terminal nucleotides of tmRNA (MutmRNA) for encoding a tag containing six histidine residues (His6tag), so as to capture and enrich the trans-translation substrates from the cell lysates through a Ni2+-NTA affinity chromatograph. The results showed that the concentrated substrates were detected as distinct and specific bands in western blotting using anti-His antibody, demonstrating that specific defective mRNAs were frequently and intensively rescued by trans-translation during the translation process in A. veronii. The substrates were analyzed by LC-MS/MS and further identified by searching a theoretically constructed database specific for A. veronii. Total of 24 potential substrates were identified, with various functions involved in metabolism, as well as structure and signal-based cellular events. Among the identified substrates, PspA and AsmA were labeled by Flag, and expressed in the presence of the modified trans-translation system in E. coli. Their labelings with MutmRNA were validated by purification through Ni2+-NTA column followed by western blotting using anti-Flag antibody. This study provided the most abundant set of endogenous targets for tmRNA in A. veronii, and facilitated further investigations about the molecular mechanism and signal pathway of tmRNA-mediated trans-translation.

中文翻译：

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大规模鉴定vermii气单胞菌中tmRNA靶向的反式翻译底物。

传递信使RNA（tmRNA）在细菌中无处不在，是反式翻译系统的核心组件，有助于标记异常合成的肽进行降解并释放停滞的核糖体。tmRNA的缺失会导致多种与细菌中重要的生理过程有关的表型。为了阐明tmRNA在水生病原菌气单胞菌中的多功能性的分子机制，我们突变了tmRNA的C末端核苷酸（MutmRNA）以编码包含六个组氨酸残基的标签（His6tag），以捕获和丰富反式翻译Ni2 + -NTA亲和色谱从细胞裂解物中提取底物。结果表明，使用抗His抗体在Western印迹中检测到浓缩的底物为不同的特异性条带，证明了在A. veronii的翻译过程中，通过转译可以频繁，密集地拯救特定的缺陷mRNA。通过LC-MS / MS对底物进行分析，并通过搜索理论上构建的针对Ver。A. veronii的数据库进一步鉴定。总共确定了24种潜在的底物，它们具有涉及代谢的各种功能，以及基于结构和信号的细胞事件。在鉴定出的底物中，PspA和AsmA用Flag标记，并在修饰的转译系统存在下在大肠杆菌中表达。通过使用Ni2 + -NTA柱纯化​​，然后使用抗Flag抗体进行Western印迹验证了MutmRNA对它们的标记。这项研究为拟南芥中的tmRNA提供了最丰富的内源性靶标，