Department of Genetics, University of Georgia, Athens, Georgia, USA.
Microbiology, University of Georgia, Athens, Georgia, USA.
Mol Microbiol. 2022 Dec;118(6):698-715. doi: 10.1111/mmi.14991. Epub 2022 Nov 5.
The alaW alaX operon encodes the Ala2 tRNAs, one of the two alanine tRNA isotypes in Escherichia coli. Our previous RNA-seq study showed that alaW alaX dicistronic RNA levels increased significantly in the absence of both RNase P and poly(A) polymerase I (PAP I), suggesting a role of polyadenylation in its stability. In this report, we show that RNase E initiates the processing of the primary alaW alaX precursor RNA by removing the Rho-independent transcription terminator, which appears to be the rate limiting step in the separation and maturation of the Ala2 pre-tRNAs by RNase P. Failure to separate the alaW and alaX pre-tRNAs by RNase P leads to poly(A)-mediated degradation of the dicistronic RNAs by polynucleotide phosphorylase (PNPase) and RNase R. Surprisingly, the thermosensitive RNase E encoded by the rne-1 allele is highly efficient in removing the terminator (>99%) at the nonpermissive temperature suggesting a significant caveat in experiments using this allele. Together, our data present a comprehensive picture of the Ala2 tRNA processing pathway and demonstrate that unprocessed RNase P substrates are degraded via a poly(A) mediated decay pathway.
alaW alaX 操纵子编码 Ala2 tRNA，是大肠杆菌中两种丙氨酸 tRNA 同工型之一。我们之前的 RNA-seq 研究表明，在缺乏 RNase P 和多聚（A）聚合酶 I（PAP I）的情况下，alaW alaX 二顺反子 RNA 水平显著增加，这表明多聚腺苷酸化在其稳定性中起作用。在本报告中，我们表明，RNase E 通过去除 Rho 非依赖性转录终止子来启动初级 alaW alaX 前体 RNA 的加工，这似乎是 RNase P 分离和成熟 Ala2 前 tRNA 的限速步骤。RNase P 未能分离 alaW 和 alaX 前 tRNA 会导致多聚腺苷酸化介导的二顺反子 RNA 被多核苷酸磷酸化酶（PNPase）和 RNase R 降解。令人惊讶的是，由 rne-1 等位基因编码的热敏性 RNase E 在非许可温度下非常有效地去除终止子（>99%），这表明在使用该等位基因的实验中存在重大问题。总之，我们的数据提供了 Ala2 tRNA 加工途径的全面描述，并表明未加工的 RNase P 底物通过多聚腺苷酸化介导的衰减途径降解。
