Singh Nongmaithem Sadananda, Varshney Umesh
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560 012, India.
Nucleic Acids Res. 2004 Nov 16;32(20):6028-37. doi: 10.1093/nar/gkh924. Print 2004.
The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on the mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. Here, we show that overexpression of tmRNA rescues the temperature-sensitive phenotype of Escherichia coli (pth(ts)). Conversely, a null mutation in ssrA enhances the temperature-sensitive phenotype of the pth(ts) strain. Consistent with our hypothesis, overexpression of tmRNA results in decreased accumulation of peptidyl-tRNA in E.coli. Furthermore, overproduction of tmRNA in E.coli strains deficient in ribosome recycling factor and/or lacking the release factor 3 enhances the rescue of pth(ts) strains. We discuss the physiological relevance of these observations to highlight a major role of tmRNA in decreasing cellular peptidyl-tRNA load.
细菌的ssrA基因编码一种具有双重功能的RNA，即tmRNA，它具有类似tRNA和类似mRNA的区域。tmRNA通过转译过程在P位点tRNA上的多肽上附加一个寡肽标签，该过程拯救了停滞在mRNA上的核糖体，并将异常蛋白质靶向降解。在细胞中，停滞核糖体的处理也由肽基-tRNA的脱落引发。连接肽与tRNA的酯键被肽基-tRNA水解酶（Pth）水解，Pth是一种必需的酶，它释放tRNA和异常肽。由于转译机制利用停滞核糖体的肽基转移酶活性来释放tRNA（与肽基-tRNA脱落相反），因此绕过了Pth回收此类tRNA的需求。因此，我们假设tmRNA可能挽救Pth的缺陷。在这里，我们表明tmRNA的过表达挽救了大肠杆菌（pth(ts)）的温度敏感表型。相反，ssrA中的无效突变增强了pth(ts)菌株的温度敏感表型。与我们的假设一致，tmRNA的过表达导致大肠杆菌中肽基-tRNA的积累减少。此外，在缺乏核糖体回收因子和/或缺乏释放因子3的大肠杆菌菌株中过量产生tmRNA增强了对pth(ts)菌株的挽救作用。我们讨论了这些观察结果的生理相关性，以突出tmRNA在降低细胞肽基-tRNA负荷中的主要作用。
