Secondary structure of the mRNA for ribosomal protein S20. Implications for cleavage by ribonuclease E.
A synthetic RNA transcript containing the entire sequence of one of the two natural mRNAs for Escherichia coli ribosomal protein S20 is a substrate for specific cleavage by an endonuclease which is or depends on ribonuclease E (Mackie, G. A. (1991) J. Bacteriol. 173, 2488-2497). Partial cleavage with ribonucleases T1 or CL3 and limited modification with dimethyl sulfate have been employed to identify residues that are likely to be single stranded in the S20 mRNA's native state. The data show that the 5' one-third of the mRNA is relatively unstructured whereas the 3' one-third is extensively folded. The latter property can account for the previously observed accumulation of a 147-residue product co-terminal with the 3' end of the S20 mRNA (Mackie, G. A. (1989) J. Bacteriol. 171, 4112-4120). Sites of cleavage by the ribonuclease E-dependent activity map to single-stranded regions of the RNA. In addition, denaturation of the RNA substrate results in loss of susceptibility to the ribonuclease E-dependent activity and simultaneous loss of the single-stranded character of the two most prominent cleavage sites. It is proposed that ribonuclease E is a single-strand-specific enzyme with few primary structural constraints but a preference for an AU dinucleotide 3' to the site of cleavage.
Department of Biochemistry, University of Western Ontario, London, Canada.
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Mackie
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014158:Transcription, Genetic
D012329:RNA, Bacterial; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C029611:ribosomal protein S20; D004722:Endoribonucleases; C022537:ribonuclease E