The study of the C-terminal region of the cyclic AMP receptor protein.
Item
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Title
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The study of the C-terminal region of the cyclic AMP receptor protein.
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Identifier
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AAI9315519
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identifier
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9315519
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Creator
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Yang, Zhaohui.
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Contributor
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Adviser: Joseph S. Krakow
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Date
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1993
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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
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Abstract
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The cAMP receptor protein, or CRP (CAP), is an allosteric protein which regulates transcription of more than 20 genes in E. coli. Transcription regulation is achieved by binding of cAMP/CRP to a specific promoter-related DNA sequence. A truncated form of CRP, CRP{dollar}\sp{lcub}\rm CY{rcub}{dollar}, was prepared by treating CRP with carboxypeptidase Y. CRP{dollar}\sp{lcub}\rm CY{rcub}{dollar}, which has lost seven C-terminal amino acids, bind cAMP with an affinity similar to that of wild-type CRP. Unlike the wild-type CRP, it is sensitive to endoproteolytic cleavage in the absence of cAMP. CRP{dollar}\sp{lcub}\rm CY{rcub}{dollar} does not bind DNA or support transcription from lac P{dollar}\sp+{dollar} by RNA polymerase. Substitution and deletion mutations at the C-terminal arginine or in the C-terminal region have been made using site-directed mutagenesis and the mutant proteins purified. The mutant CRPs bound cAMP with an affinity similar to that of the wild-type CRP. The cleavage patterns of the mutant proteins by the proteases were similar to those of the wild-type CRP. Some of the mutants showed weak binding to a DNA fragment containing the lac P{dollar}\sp+,{dollar} while others bound with affinities similar to wild-type CRP. DNase I footprinting showed that binding to lac P{dollar}\sp+{dollar} by some of the mutant CRPs was enhanced in the presence of RNA polymerase, suggesting that there was interaction between the two proteins and that such interaction stabilized the binding of both proteins to the DNA. The mutant proteins generally retained 15 to 30% activity of the wild-type CRP in supporting abortive initiation from lac P{dollar}\sp+{dollar} by RNA polymerase. The results show that the C-terminal arginine as well as the other amino acids studied in the C-terminal region play a role in the function of CRP.
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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.
