EFFECTS OF NUCLEOTIDE-SEQUENCE ON THE SPECIFICITY OF RNE-DEPENDENT AND RNASE E-MEDIATED CLEAVAGES OF RNA I-ENCODED BY THE PBR322 PLASMID

RNase E, an endoribonuclease encoded by the Escherichia coil ams/rne/h mp1 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 I half-life in cells cultured at a n elevated temperature and imparts temperature sensitivity on RNase E isolated from the mutant strain. Here we report the effects of specifi c sequence changes introduced by site-directed mutagenesis on the loca tion of ribonucleolytic cleavage near the 5' end of pBR322 RNA I in rn e-3071 and congenic rne(+) E. coil and on cleavage of RNA I by RNase E in vitro. Primer extension analyses showed that the occurrence and po sition of cleavages in vivo and in vitro are altered highly specifical ly by sequence changes but that the site of cleavage bears no simple r elationship to a particular nucleotide order. Our results do not suppo rt either the notion that cleavage by RNase E is determined by a conse nsus sequence or the contrary view that RNase E is a virtually nonspec ific single-stranded endonuclease with a preference for cutting 5' to an AU dinucleotide.