A-CLEAVAGE(U CONTENT RATHER THAN A PARTICULAR NUCLEOTIDE ORDER DETERMINES THE SPECIFICITY OF RNASE E)

Ribonuclease E has a central role in Escherichia coil mRNA decay and i s dependent on a functional product of the me (also called ams or hmp1 ) gene. We investigated the requirements for RNase E cleavage by intro ducing random mutations into the decanucleotide region at the 5' end o f pACYC184 RNA I and studying the effects of these mutations on the po sition of rne-dependent cleavage in vivo and RNase E-mediated cutting in vitro. We find that the precise point of RNase E cleavage can be al tered specifically and reproducibly by sequence changes in the region cleaved and, therefore, is not determined by a distance measured in nu cleotides from any other sequence or region of secondary structure in RNA I. Although cleavage by RNase E occurs within sequences rich in A and/or U nucleotides and is affected by the extent of continuity of A and U nucleotides in the regions cleaved, there is no simple relations hip between the order of nucleotides and the phosphodiester bond cleav ed. Thus, our results are not consistent with either the notion that R Nase E cleavages are determined by a simple consensus sequence or the contrary view that RNase E has few primary structural constraints othe r than a preference for cleaving 5' to an AU dinucleotide.