Department of Microbiology, Immunobiology and Genetics, Max Perutz Labs, University of Vienna, Vienna Biocenter (VBC), Vienna, Austria.
Doctoral School in Microbiology and Environmental Science, University of Vienna, Vienna, Austria.
Methods Mol Biol. 2024;2741:255-272. doi: 10.1007/978-1-0716-3565-0_14.
Bacterial small RNAs (sRNAs) can be equipped at the 5' end with triphosphate (5'PPP) or monophosphate (5'P) groups, depending on whether they are primary transcripts, undergo dephosphorylation or originate via processing. Often, 5' groups hallmark RNAs for rapid decay, but whether this also applies to sRNAs is little explored. Moreover, the sRNA 5'P group could activate endoribonuclease RNase E to cleave the base-paired target RNA, but a tool for investigation in vivo was lacking. Here, we describe a two-plasmid system suitable for the generation of 5' monophosphorylated RNAs on demand inside the cell. The sRNA gene of interest is fused to the 3' end of a fragment of sRNA GlmZ and transcribed from a plasmid in an IPTG-inducible manner. The fusion RNA gets cleaved upon arabinose-controlled expression of rapZ, provided on a compatible plasmid. Adaptor protein RapZ binds the GlmZ aptamer and directs RNase E to release the sRNA of choice with 5'P ends. An isogenic plasmid generating the same sRNA with a 5'PPP end allows for direct comparison. The fates of the sRNA variants and target RNA(s) are monitored by Northern blotting. This tool is applicable to E. coli and likely other enteric bacteria.
细菌小 RNA (sRNA) 可以在 5' 端配备三磷酸 (5'PPP) 或单磷酸 (5'P) 基团，具体取决于它们是初级转录物、经历去磷酸化还是通过加工产生。通常，5' 基团标志着 RNA 快速降解，但这是否也适用于 sRNA 则研究甚少。此外，sRNA 的 5'P 基团可以激活内切核酸酶 RNase E 来切割碱基配对的靶 RNA，但缺乏体内研究的工具。在这里，我们描述了一种适用于在细胞内按需生成 5' 单磷酸化 RNA 的两质粒系统。感兴趣的 sRNA 基因融合到 sRNA GlmZ 的 3' 端，并从质粒中以 IPTG 诱导的方式转录。融合 RNA 在阿拉伯糖控制的 rapZ 表达时被切割，rapZ 提供在兼容的质粒上。衔接蛋白 RapZ 结合 GlmZ 适体并指导 RNase E 释放带有 5'P 末端的所选 sRNA。生成具有 5'PPP 末端的相同 sRNA 的同基因质粒允许直接比较。sRNA 变体和靶 RNA(s) 的命运通过 Northern 印迹监测。该工具适用于大肠杆菌和可能的其他肠道细菌。
