SITE-SPECIFIC RNASE-E CLEAVAGE OF OLIGONUCLEOTIDES AND INHIBITION BY STEM-LOOPS

THE enzyme RNase E (ref. 1) cuts RNA at specific sites within single-s tranded segments(2-6). The role of adjacent regions of secondary struc ture in such cleavages is controversial(7-10). Here we report that 10- 13-nucleotide oligomers lacking any stem-loop but containing the RNase E-cleaved sequence of RNA I11-13, the antisense repressor of replicat ion of ColE1-type plasmids, are cut at the same phosphodiester bond as , and 20 times more efficiently than, RNA I. These findings indicate t hat, contrary to previous proposals(8,9), stem-loops do not serve as e ntry sites for RNase E, but instead limit cleavage at potentially susc eptible sites. Cleavage was reduced further by mutations in a non-adja cent stem-loop, suggesting that distant conformational changes can als o affect enzyme access. Modulation of RNase E cleavages by stem-loop r egions and to a lesser extent by higher-order structure may explain wh y this enzyme, which does not have stringent sequence specificity(6,12 ,13), cleaves complex RNAs at a limited number of sites.