Evidence for helical unwinding of an RNA substrate by the RNA enzyme RNase P: use of an interstrand disulfide crosslink in substrate.
To gain an understanding of structural changes induced in substrates by Escherichia coli ribonuclease P (RNase P), we have incorporated an interstrand disulfide crosslink proximal to the cleavage site in a model substrate. RNase P is able to process the reduced, non-crosslinked form of this substrate as well as a substrate in which the free thiol molecules have been alkylated with iodoacetamide. However, the oxidized, crosslinked form is cleaved at a significantly lower rate. Therefore, helical unwinding of the analog of the aminoacyl stem of the substrate near its site of cleavage may be necessary for efficient processing by E. coli RNase P.
Pomeranz Krummel
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Department of Molecular Cellular and Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, 06511, USA.
Kent
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MacMillan
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Altman
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D000477:Alkylating Agents / Q000378:metabolism; D000478:Alkylation; D020029:Base Pairing / Q000235:genetics*; D001483:Base Sequence; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D020035:Holoenzymes / Q000378:metabolism; D007460:Iodoacetamide / Q000378:metabolism; D007700:Kinetics; D008957:Models, Genetic; D008958:Models, Molecular; D010084:Oxidation-Reduction; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000378:metabolism*; D012331:RNA, Fungal / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
D000477:Alkylating Agents; D004220:Disulfides; D029968:Escherichia coli Proteins; D020035:Holoenzymes; D016337:RNA, Catalytic; D012331:RNA, Fungal; D012360:RNA, Transfer, Phe; D013438:Sulfhydryl Compounds; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D007460:Iodoacetamide