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-stranded segments. The role of adjacent regions of secondary structure in such cleavages is controversial. Here we report that 10-13-nucleotide oligomers lacking any stem-loop but containing the RNase E-cleaved sequence of RNA I, the antisense repressor of replication of ColE1-type plasmids, are cut at the same phosphodiester bond as, and 20 times more efficiently than, RNA I. These findings indicate that, contrary to previous proposals, stem-loops do not serve as entry sites for RNase E, but instead limit cleavage at potentially susceptible sites. Cleavage was reduced further by mutations in a non-adjacent stem-loop, suggesting that distant conformational changes can also affect enzyme access. Modulation of RNase E cleavages by stem-loop regions and to a lesser extent by higher-order structure may explain why this enzyme, which does not have stringent sequence specificity, cleaves complex RNAs at a limited number of sites.
McDowall
K J
KJ
Department of Genetics, Stanford University School of Medicine, California 94305.
Kaberdin
V R
VR
Wu
S W
SW
Cohen
S N
SN
Lin-Chao
S
S
D001483:Base Sequence; D004722:Endoribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006163:Ribonuclease T1 / Q000378:metabolism; D013379:Substrate Specificity
D009843:Oligoribonucleotides; C042027:RNA I; D012329:RNA, Bacterial; D004722:Endoribonucleases; D006163:Ribonuclease T1; C022537:ribonuclease E