Effects of nucleotide sequence on the specificity of rne-dependent and RNase E-mediated cleavages of RNA I encoded by the pBR322 plasmid.

Abstract

RNase E, an endoribonuclease encoded by the Escherichia coli ams/rne/hmp1 locus, cleaves RNA I, an antisense regulator of the replication of ColE1 type plasmids, in a single-stranded region near its 5' end. The rne-3071 mutation prolongs the RNA 1 half-life in cells cultured at an elevated temperature and imparts temperature sensitivity on RNase E isolated from the mutant strain. Here we report the effects of specific sequence changes introduced by site-directed mutagenesis on the location of ribonucleolytic cleavage near the 5' end of pBR322 RNA I in rne-3071 and congenic rne+ E. coli and on cleavage of RNA I by RNase E in vitro. Primer extension analyses showed that the occurrence and position of cleavages in vivo and in vitro are altered highly specifically by sequence changes but that the site of cleavage bears no simple relationship to a particular nucleotide order. Our results do not support either the notion that cleavage by RNase E is determined by a consensus sequence or the contrary view that RNase E is a virtually nonspecific single-stranded endonuclease with a preference for cutting 5' to an AU dinucleotide.