Genetic uncoupling of the dsRNA-binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III -: the effect of dsRNA binding on gene expression
Genetic uncoupling of the dsRNA-binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III -: the effect of dsRNA binding on gene expression
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DOI：
10.1046/j.1365-2958.1998.00828.x
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发表时间：
1998-05-01
影响因子：
3.6
通讯作者：
Court, DL
中科院分区：
文献类型：
--
作者：
Dasgupta, S;Fernandez, L;Court, DL
RNase III是一种双链RNA特异性核酸内切酶，由于其能够通过影响mRNA稳定性或mRNA翻译效率的转录后控制来影响大量无关基因的表达，因此被认为是大肠杆菌的全局调节因子之一。在这里，我们描述了携带点突变的细菌的表型在我，编码RNase III的基因。通过测量已知RNase III调节基因的表达，在体内分析了突变蛋白的底物识别和RNA加工特性，并在体外分析了蛋白对已知双链RNA底物的结合和切割活性。我们的结果表明，尽管点突变rnc 70表现出所有常见的类基因表型，但与其他突变不同，它对野生型等位基因是显性的，rnc 70的多拷贝表达可以抑制野生型me等位基因在一定遗传背景下的致死表型;它还可以通过竞争野生型内切核酸酶的RNA结合位点来抑制RNase III介导的λ N基因翻译的激活。该突变蛋白在体外不能切割标准RNase III底物，但当通过poly（rl）：poly（rC）柱时表现出对双链RNA的亲和力。过滤器结合和凝胶迁移分析与纯化的Rnc 70表明，突变蛋白结合到已知的RNase III mRNA底物的位点特异性的方式。纯化的酶和标记的RNA在体外处理反应表明，在体内的突变体酶在野生型的显性效应不一定是由混合二聚体的形成。因此，rnc 70突变产生的突变体RNase III具有受损的核酸内切活性，但不阻断其识别和结合双链RNA底物的能力。
RNase III, a double-stranded RNA-specific endonuclease, is proposed to be one of Escherichia coli's global regulators because of its ability to affect the expression of a large number of unrelated genes by influencing post-transcriptional control of mRNA stability or mRNA translational efficiency. Here, we describe the phenotypes of bacteria carrying point mutations in me, the gene encoding RNase III. The substrate recognition and RNA-processing properties of mutant proteins were analysed in vivo by measuring expression from known RNase III-modulated genes and in vitro from the proteins' binding and cleavage activities on known double-stranded RNA substrates, Our results show that although the point mutation rnc70 exhibited all the usual me null-like phenotypes, unlike other mutations, it was dominant over the wild-type allele, Multicopy expression of rnc70 could suppress a lethal phenotype of the wild-type me allele in a certain genetic background; it could also inhibit the RNase III-mediated activation of lambda N gene translation by competing for the RNA-binding site of the wild-type endonuclease. The mutant protein failed to cleave the standard RNase III substrates in vitro but exhibited an affinity for double-stranded RNA when passed through poly(rl):poly(rC) columns. Filter binding and gel-shift assays with purified Rnc70 showed that the mutant protein binds to known RNase III mRNA substrates in a site-specific manner. In vitro processing reactions with purified enzyme and labelled RNA showed that the in vivo dominant effect of the mutant enzyme over the wild-type was not necessarily caused by formation of mixed dimers. Thus, the rnc70 mutation generates a mutant RNase III with impaired endonucleolytic activity but without blocking its ability to recognize and bind double-stranded RNA substrates.