Monoclonal antibodies directed against alpha subunit as probes of the structure and function of Escherichia coli RNA polymerase.
Item
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Title
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Monoclonal antibodies directed against alpha subunit as probes of the structure and function of Escherichia coli RNA polymerase.
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Identifier
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AAI9000062
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identifier
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9000062
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Creator
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Riftina, Faina.
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Contributor
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Adviser: Joseph S. Krakow
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Date
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1989
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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 | Biology, Molecular
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Abstract
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Anti-alpha monoclonal antibodies (mAbs) have been used to study topological arrangements of the two alpha subunits in E. coli RNA polymerase and to elucidate the role of the alpha subunit in transcription initiation. None of the studied anti-alpha mAbs inhibit the d(A-T){dollar}\sb{lcub}\rm n{rcub}{dollar}-directed synthesis of r(A-U){dollar}\sb{lcub}\rm n{rcub}{dollar}. mAb 126C6 strongly inhibits cAMP-CRP-dependent abortive initiation with lac P+ and partially inhibits abortive initiation with lac L8UV5 promoter; mAb 129C4 and mAb 124D1 are without effect. Kinetic analysis of open complex formation of mAb 126C6-polymerase with lac L8UV5 showed that both binding and isomerization steps are affected. DNase I footprinting and protection methylation studies indicate that non-optimal contacts are established between mAb 126C6-polymerase and lac UV5 promoter. The data suggest that for mAb 126C6-polymerase the rate-limiting step in the open promoter complex formation is the proper alignment of the DNA with respect to the catalytic site of the enzyme. Analysis of the DNase I footprints of the mAb 126C6-polymerase complexed with cAMP-CRP-lac P+ suggests that interactions between CRP and polymerase are affected by binding of the mAb to polymerase.;Effects of the mAbs on reassembly of core enzyme from the subunit mixture suggest that at least one of the alpha subunits undergoes conformational changes during core enzyme assembly. The increase in the affinity of mAb 126C6 for assembled alpha compared to free alpha supports this conclusion.;Double antibody binding studies show that the epitopes for the non-inhibitory mAbs are available on only one of the alpha subunits in RNA polymerase and the epitope on each alpha subunit is available for binding by the inhibitory mAb 126C6. The studies also suggest that in the holoenzyme the sigma subunit is positioned close to one of the alpha subunits. Double antibody binding studies indicate that the alpha-beta, but not alpha{dollar}\sb2{dollar}beta, complex is the minimal stable subassembly, suggesting that the in vitro assembly of RNA polymerase core enzyme may proceed via the formation of a more stable alpha-beta complex followed by addition of the second alpha subunit.
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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.
