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dc.contributor.authorWang, Ming Jing
dc.contributor.authorGegenheimer, Peter Albert
dc.identifier.citationWang, Ming Jing, and Peter Gegenheimer. "Substrate Masking: Binding of RNA by EGTA-inactivated Micrococcal Nuclease Results in Artifactual Inhibition of RNA Processing Reactions." Nucleic Acids Research 18.22 (1990): 6625-631.
dc.descriptionThis is the published version. Copyright Oxford University Press 1991. All rights reserved.en_US
dc.description.abstractInhibition of an RNA processing reaction after treatment with the Ca2+- dependent micrococcal nuclease (MN) is often used as a criterion for the presence of a required RNA or ribonucleoproteln component in the system. Following MN digestion, the nuclease is inactivated with EGTA and radiolabeled substrate is added to assay for remaining RNA processing activity. We found previously that inhibition of RNA processing by MN need not involve RNA hydrolysis: EGTA-inactivated MN can suppress RNA processing if the assay is performed in the absence of carrier RNA. We now demonstrate both by native gel electrophoresis and by nitrocellulose filter retention that EGTA-inactivated MN forms a complex with free RNA which can be dissociated by addition of synthetic polynucleotldes or heparin. In the absence of Ca2+ , nuclease binds to precursor tRNA with an apparent KD ≃ 1.4 × 10−6 M, comparable to its reported affinity for DNA. In an assay for endonucleolytlc tRNA maturation, inactivated MN bound to radiolabeled pre-tRNA physically blocks the sites of endonuclease cleavage and prevents tRNA processing. We call this phenomenon ‘substrate masking’. Addition of excess carrier RNA competes with pre-tRNA for MN binding and restores normal processing.en_US
dc.publisherOxford University Pressen_US
dc.titleSubstrate masking: binding of RNA by EGTA-inactivated micrococcal nuclease results in artifactual inhibition of RNA processing reactionsen_US
kusw.kuauthorGegenheimer, Peter Albert
kusw.kudepartmentMolecular Biosciencesen_US
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kusw.oaversionScholarly/refereed, publisher version
kusw.oapolicyThis item does not meet KU Open Access policy criteria.

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