Modulation of the activity of RNase E in vitro by RNA sequences and secondary structures 5' to cleavage sites.
The endoribonuclease RNase E is believed to initiate the degradation of many mRNAs in Escherichia coli, yet the mechanism by which it recognizes cleavage sites is poorly understood. We have prepared derivatives of the mRNA encoding ribosomal protein S20 which contain a single major RNase E cleavage site at residues 300/301 preceded by variable 5' extensions. Three of these RNAs are cleaved in vitro with significantly reduced efficiencies relative to the intact S20 mRNA by both crude RNase E and pure Rne protein (endonuclease component of RNase E). In all three substrates as well as in the full-length mRNA the major cleavage site itself remains single-stranded. One such substrate (t84D) contains a 5' stem-loop structure characterized by three noncanonical A-G pairs. Removal or denaturation of the stem restores efficient cleavage at the major RNase E site. The other two contain single-stranded 5'-termini but apparently lack cleavage sites near the termini. Our data show that sensitivity to RNase E can be influenced by distant structural motifs in the RNA and also suggest a model in which the initial recognition and cleavage of a substrate near its 5' end facilitates sequential cleavages at more distal sites. The model implies that RNase E contains at least a dimer of the Rne subunit and that the products of the first cleavage are retained by Rne prior to the second cleavage.
Mackie
G A
GA
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada. gamackie@unixg.ubc.ca
Genereaux
J L
JL
Masterman
S K
SK
D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C029611:ribosomal protein S20; D004722:Endoribonucleases; C022537:ribonuclease E