Antibodies as probes of the structure and function of the alpha and beta' subunits of the Escherichia coli RNA polymerase.
Item
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Title
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Antibodies as probes of the structure and function of the alpha and beta' subunits of the Escherichia coli RNA polymerase.
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Identifier
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AAI9618102
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identifier
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9618102
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Creator
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Sharif, Karim Ahmad.
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Contributor
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Adviser: Joseph S. Krakow
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Date
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1996
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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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Biology, Molecular | Biology, Genetics
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Abstract
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The epitopes have been localized for a set of monoclonal antibodies specific for the {dollar}\alpha{dollar} subunit of the E. coli RNA polymerase. These antibodies were also used to investigate the role of {dollar}\alpha{dollar} in transcription activation with CRP dependent as well as activator independent promoters. Immunogenic regions representing the surfaces that may be exposed on {dollar}\alpha{dollar} have been assessed by using polyclonal antibodies specific to shorter {dollar}\alpha{dollar} fragments. Subunit specific monoclonal antibodies have also been used to assess {dollar}\sigma{dollar} interaction with the individual subunits of RNA polymerase.;Anti-{dollar}\alpha{dollar} monoclonal antibodies studied are classified into 3 groups based on their epitopic assignments. Group 1 mAb 123C2 maps in the N-terminus of {dollar}\alpha{dollar} between amino acids 1-23; Group 2 antibodies (mAb 129C4, mAb 124D1 and mAb 121C5) map in the central region between amino acids 190-210; Group 3 antibodies (mAb 130B1 and mAb 225C6) map in the C-terminus between amino acids 310-320. mAb 130C2 is anomalous since it maps to the N-terminus between amino acids 1-23 as well as the C-terminus between amino acids 320-329. Three antibodies (mAb 130C2, mAb 121C5, and mAb 125C6) inhibited CRP-dependent initiation with lac P{dollar}\sp+{dollar} but not with lac UV5 or gal P{dollar}\sp+.{dollar} Inhibition was observed with free RNA polymerase and RP{dollar}\sb{lcub}\rm c{rcub}{dollar}; the preformed RP{dollar}\sb{lcub}\rm o{rcub}{dollar} was insensitive. Only lac P{dollar}\sp+{dollar} was sensitive to these anti-{dollar}\alpha{dollar} antibodies supporting the concept that the mode of interaction of RNA polymerase with CRP differs between lac P{dollar}\sp+{dollar} and gal P{dollar}\sp+.{dollar} Anti-{dollar}\alpha{dollar} antibodies failed to inhibit transcription from several activator independent promoters.;The immunogenic regions on {dollar}\alpha{dollar}, revealed by polyclonal antibodies, mostly correspond to the regions involved in either subunit-subunit contacts or molecular interactions with cis-acting UP elements or trans-acting transcription factors. The determined regions also are in good agreement with the epitopes identified for the monoclonal antibodies.;Subunit specific monoclonal antibodies were used to assess interaction between the individual subunits and {dollar}\sigma\sp{lcub}70{rcub}{dollar} by the ability of another polymerase subunit to coimmunoprecipitate {dollar}\sigma{dollar}. The results indicate that free {dollar}\beta\sp\prime{dollar} can bind to {dollar}\sigma{dollar} in solution in the presence or absence of heparin, tRNA or nonspecific DNA. This was also confirmed by HPLC gel filtration. This interaction was mapped to the N-terminal domain of {dollar}\beta\sp\prime{dollar} including residues 201-477. None of the other free subunits or the {dollar}\alpha\sb2\beta{dollar} complex coimmunoprecipitated the {dollar}\sigma{dollar} 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.
