Department of Genetics, School of Medicine, Stanford University, Stanford, California, USA.
J Bacteriol. 2012 Apr;194(8):1919-26. doi: 10.1128/JB.06652-11. Epub 2012 Feb 10.
Escherichia coli cells normally require RNase E activity to propagate and form colonies. Using random Tn10 insertion mutagenesis, we screened for second-site suppressor mutations that restore colony-forming ability (CFA) to E. coli cells lacking RNase E function and found mutations in three separate chromosomal loci that had this phenotype. Restoration of CFA by mutations in two of the genes identified was observed only in nutrient-poor medium, whereas the effects of mutation of the ATP-dependent RNA helicase DeaD were medium independent. Suppression of the rne mutant phenotype by inactivation of deaD was partial, as rne deaD doubly mutant bacteria had a greatly prolonged generation time and grew as filamentous chains in liquid medium. Moreover, we found that CFA restoration by deaD inactivation requires normal expression of the endogenous rng gene in doubly mutant rne deaD cells. Second-site suppression by deaD mutation was attributable specifically to ablation of the helicase activity of DeaD and was reversed by adventitious expression of RhlE or RNase R, both of which can unwind double-stranded RNA. Our results suggest a previously unsuspected role for RNA secondary structure as a determinant of RNase E essentiality.
大肠杆菌细胞通常需要 RNase E 活性来繁殖和形成菌落。使用随机 Tn10 插入诱变，我们筛选了能够恢复缺乏 RNase E 功能的大肠杆菌细胞集落形成能力 (CFA) 的第二位置抑制突变，并在三个独立的染色体位置发现了具有这种表型的突变。在两种鉴定的基因中的突变恢复 CFA 仅在营养贫乏的培养基中观察到，而 ATP 依赖性 RNA 解旋酶 DeaD 的突变的影响是培养基独立的。deaD 的失活对 rne 突变表型的抑制是部分的，因为 rne deaD 双重突变细菌的代时大大延长，并在液体培养基中以丝状链的形式生长。此外，我们发现，deaD 失活导致的 CFA 恢复需要在双重突变 rne deaD 细胞中内源 rng 基因的正常表达。deaD 突变的第二位置抑制归因于 DeaD 解旋酶活性的特异性缺失，并通过意外表达 RhlE 或 RNase R 逆转，这两者都可以展开双链 RNA。我们的结果表明，RNA 二级结构作为 RNase E 必需性决定因素的作用以前是未知的。
