Ehretsmann C P, Carpousis A J, Krisch H M
Department of Molecular Biology, University of Geneva, Switzerland.
Genes Dev. 1992 Jan;6(1):149-59. doi: 10.1101/gad.6.1.149.
Endoribonuclease RNase E has an important role in the processing and degradation of bacteriophage T4 and Escherichia coli mRNAs. We have undertaken a mutational analysis of the -71 RNase E processing site of T4 gene 32. A series of mutations were introduced into a synthetic T4 sequence cloned on a plasmid, and their effects on processing were analyzed in vivo. The same mutations were transferred into T4 by homologous recombination. In both the plasmid and the phage contexts the processing of the transcripts was similarly affected by the mutations. Partially purified RNase E has also been used to ascertain the effect of these mutations on RNase E processing in vitro. The hierarchy of the efficiency of processing of the various mutant transcripts was the same in vivo and in vitro. These results and an analysis of all of the known putative RNase E sites suggest a consensus sequence RAUUW (R = A or G; W = A or U) at the cleavage site. Modifications of the stem-loop structure downstream of the -71 site indicate that a secondary structure is required for RNase E processing. Processing by RNase E was apparently inhibited by sequences that sequester the site in secondary structure.
核糖核酸内切酶RNase E在噬菌体T4和大肠杆菌mRNA的加工与降解过程中发挥着重要作用。我们对T4基因32的-71 RNase E加工位点进行了突变分析。将一系列突变引入克隆在质粒上的合成T4序列中，并在体内分析它们对加工的影响。通过同源重组将相同的突变导入T4。在质粒和噬菌体环境中，转录本的加工受到突变的类似影响。部分纯化的RNase E也被用于确定这些突变对体外RNase E加工的影响。各种突变转录本的加工效率等级在体内和体外是相同的。这些结果以及对所有已知假定RNase E位点的分析表明，切割位点存在一个共有序列RAUUW（R = A或G；W = A或U）。-71位点下游茎环结构的修饰表明，RNase E加工需要二级结构。RNase E的加工显然受到将该位点隔离在二级结构中的序列的抑制。
