Studies on the structure and function of the beta-prime subunit of Escherichia coli RNA polymerase.
Item
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Title
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Studies on the structure and function of the beta-prime subunit of Escherichia coli RNA polymerase.
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Identifier
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AAI9304697
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identifier
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9304697
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Creator
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Luo, Jianying.
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Contributor
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Adviser: Joseph S. Krakow
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Date
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1992
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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 | Biology, Microbiology
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Abstract
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The E. coli RNA polymerase is a multisubunit enzyme. The holoenzyme active during initiation has the structure: {dollar}\alpha\sb2\beta\beta\sp\prime\sigma{dollar} while the catalytic competent core enzyme has the structure: {dollar}\alpha\sb2\beta\beta\sp\prime{dollar}. Monoclonal antibodies (mAbs) raised against the {dollar}\beta\sp\prime{dollar} subunit of the E. coli RNA polymerase were used to probe the structure and function of this subunit. Of the five anti-{dollar}\beta\sp\prime{dollar} monoclonal antibodies studied only mAb 311G2 is a strong inhibitor of RNA polymerase activity. This antibody binds to an epitope which is exposed in both the assembled holoenzyme and isolated {dollar}\beta\sp\prime{dollar} subunit. In contrast, the null antibodies bind to the free {dollar}\beta\sp\prime{dollar} subunit but very weakly to native RNA polymerase. It would appear that the {dollar}\beta\sp\prime{dollar} domain in which their epitopes reside is either conformationally altered or blocked due to interaction with other subunits in native RNA polymerase. In order to locate the positions of the epitopes for these 5 monoclonal antibodies, a series of overlapping deletion mutants has been constructed by partial restriction and religation of the {dollar}\beta\sp\prime{dollar} gene present in pT7{dollar}\beta\sp\prime{dollar} (Zalenskaya et al., Gene 89 (1990) 7-12). The presence of the epitopes for each of the anti-{dollar}\beta\sp\prime{dollar} monoclonal antibodies was assessed by Western blotting. The results indicate that the epitopes for mAb 340F11, mAb 370F3, mAb 371D6, and mAb 372B2 are located between amino acids 817 to 876. This region may be important in subunit-subunit interaction. The epitope for the inhibitory antibody, mAb 311G2, is located between amino acids 1047 to 1093. This region may be involved in the catalytic function of RNA polymerase.;Truncated {dollar}\beta\sp\prime{dollar} mutant proteins were also used to map the region on {dollar}\beta\sp\prime{dollar} involved in the assembly of RNA polymerase. Results suggest that the N-terminal region of {dollar}\beta\sp\prime{dollar} is involved in the assembly of core enzyme, and a region between aa 201 to 477 on {dollar}\beta\sp\prime{dollar} may be involved in the interaction between the {dollar}\beta\sp\prime{dollar} subunit and 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.
