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    Substrate masking: binding of RNA by EGTA-inactivated micrococcal nuclease results in artifactual inhibition of RNA processing reactions

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    Issue Date
    1991
    Author
    Wang, Ming Jing
    Gegenheimer, Peter Albert
    Publisher
    Oxford University Press
    Type
    Article
    Article Version
    Scholarly/refereed, publisher version
    Metadata
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    Abstract
    Inhibition 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.
    Description
    This is the published version. Copyright Oxford University Press 1991. All rights reserved.
    URI
    http://hdl.handle.net/1808/17354
    DOI
    https://doi.org/10.1093/nar/18.22.6625
    Collections
    • Molecular Biosciences Scholarly Works [590]
    Citation
    Wang, 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. http://dx.doi.org/10.1093/nar/18.22.6625.

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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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