Cormack R S, Mackie G A
Department of Biochemistry, University of Western Ontario, London, Canada.
J Mol Biol. 1992 Dec 20;228(4):1078-90. doi: 10.1016/0022-2836(92)90316-c.
Processing of 9 S precursor RNA in Escherichia coli requires the endoribonuclease RNase E, which makes two cleavages to liberate p5, the immature form of 5 S rRNA. The contributions of primary and secondary structure to RNase E-mediated cleavage of 9 S RNA were investigated. The structure of the 5' domain of 9 S RNA was probed by partial ribonuclease digestion and chemical modification. Our structural analysis of 9 S RNA supports a model in which the 5' spacer domain folds into tandem hairpins so that the first processing cleavage site 5' to the 5 S moiety resides in a stretch of single-stranded residues. Site-directed mutagenesis of a cloned 9 S RNA sequence was performed and synthetic transcripts derived from a variety of such mutant templates were assayed as substrates for RNase E-dependent endonuclease activity in fractionated extracts. Partial or complete deletion of the 5 S sequence did not eliminate site-specific processing of 9 S RNA. Mutations affecting the 5' domain revealed that secondary structure upstream from the first cleavage site is important in maintaining efficient processing. However, secondary structure downstream from either cleavage site is dispensable. Our results suggest that RNase E specifically recognizes and cleaves single-stranded RNA sequences only when presented in a proper conformational context. Adjacent secondary structures appear to play a direct and critical role in the enzyme's recognition of its substrate. Additionally, it may serve to anchor single-stranded regions to ensure the availability of the RNase E cleavage sites.
大肠杆菌中9S前体RNA的加工需要核糖核酸内切酶RNase E，它进行两次切割以释放p5，即5S rRNA的未成熟形式。研究了一级和二级结构对RNase E介导的9S RNA切割的作用。通过部分核糖核酸酶消化和化学修饰探测了9S RNA 5'结构域的结构。我们对9S RNA的结构分析支持这样一个模型，即5'间隔区结构域折叠成串联发夹结构，使得5S部分5'端的第一个加工切割位点位于一段单链残基中。对克隆的9S RNA序列进行了定点诱变，并将来自各种此类突变模板的合成转录本作为底物，在分级分离的提取物中检测其对依赖RNase E的内切核酸酶活性。5S序列的部分或完全缺失并未消除9S RNA的位点特异性加工。影响5'结构域的突变表明，第一个切割位点上游的二级结构对于维持高效加工很重要。然而，任何一个切割位点下游的二级结构都是可有可无的。我们的结果表明，RNase E仅在适当的构象环境中才特异性识别并切割单链RNA序列。相邻的二级结构似乎在酶对其底物的识别中起直接和关键的作用。此外，它可能有助于锚定单链区域，以确保RNase E切割位点的可用性。
