CHARACTERIZATION OF EFFECTS OF ANTI-BETA AND ANTI-BETA' MONOCLONAL ANTIBODIES ON THE ACTIVITY OF THE RNA POLYMERASE FROM ESCHERICHIA COLI (ENZYME INHIBITOR).
Item
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Title
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CHARACTERIZATION OF EFFECTS OF ANTI-BETA AND ANTI-BETA' MONOCLONAL ANTIBODIES ON THE ACTIVITY OF THE RNA POLYMERASE FROM ESCHERICHIA COLI (ENZYME INHIBITOR).
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Identifier
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AAI8629731
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identifier
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8629731
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Creator
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ROCKWELL, PATRICIA.
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Contributor
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Joseph Krakow
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Date
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1986
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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Monoclonal antibodies (mAbs) directed against antigenic determinants on the beta and beta' subunits of the E. coli RNA polymerase were prepared using purified subunits as the immunogens. Anti-beta and anti-beta' mAbs were found which inhibited polymerase activity. Inhibition by the anti-beta mAb 210E8 varied with template sequence and conformation while the anti-beta' mAb 311G2 exhibited a potent inhibition on all templates studied. The abortive initiation reaction was more greatly affected by mAb 210E8 on linear than on supercoiled templates. On the supercoiled TAC16 promoter inhibition by mAb 210E8 was relieved when the spacer length between the -10 and -35 consensus regions was shifted from 16 to 18 base pairs. On supercoiled lac UV5 mAb 210E8 did not change the rate at which polymerase formed the closed promoter complex but decreased the rate of isomerization to form the open complex.;The reactivity of polymerase-lac UV5 (and TAC16) promoter complexes with DNase I and dimethyl sulfate was modified by the inhibitory mAbs. The mAb 210E8-polymerase-lac UV5 promoter complex forms an unstable intermediate that could not convert to a fully active RP(,o). Anti-beta' mAb 311G2-polymerase-promoter complexes are stable but inactive.;The data suggest that the beta and beta' subunits perform different functions. Evidence is provided supporting the role of the beta subunit in catalysis and in the positioning of polymerase on the promoter preceding RP(,o) formation. The beta' subunit is also implicated in catalysis in addition to its role in template bindings. The studies demonstrate that subunit-specific monoclonal antibodies are novel reagents with which to probe the relation of structure and function in RNA polymerase.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biology
