BIOCHEMICAL STUDIES ON THE DNA BINDING FUNCTION OF THE CYCLIC-AMP RECEPTOR PROTEIN OF ESCHERICHIA COLI.
Item
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Title
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BIOCHEMICAL STUDIES ON THE DNA BINDING FUNCTION OF THE CYCLIC-AMP RECEPTOR PROTEIN OF ESCHERICHIA COLI.
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Identifier
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AAI8611322
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identifier
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8611322
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Creator
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ANGULO, JESUS ALEXANDER.
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Contributor
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Joseph S. 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 | Biology, Microbiology
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Abstract
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The cAMP receptor protein (CRP) is an allosteric protein in which binding of cAMP effects a conformational change with a consequent increased affinity for DNA. Binding of double-stranded deoxyribopolynucleotides and calf thymus DNA by cAMP-CRP confers protection against attack by trypsin, subtilisin, Staph. aureus V8 protease and clostripain. Of the single-stranded deoxy- and ribopolynucleotides tested, only r(I)(,n) and r(A)(,n) gave significant protection against attack by these proteases. Since the cutting sites for trypsin (Lys 130) and subtilisin (Leu 116) are not part of the C-terminal DNA binding domain, it would appear that binding of DNA may confer conformational changes on other regions of cAMP-CRP.;In the absence of cAMP, CRP is resistant to proteolysis. Incubation of CRP-DNA with trypsin results in the accumulation of two novel fragments. CRP-DNA is partially sensitive to digestion by chymotrypsin but resistant to attack by subtilisin, the Staph. aureus V8 protease and clostripain. Cleavage of CRP-DNA to fragments is accompanied by the loss of ('3)H-cAMP binding activity. All double-stranded deoxyribopolynucleotides tested confer a conformation on CRP which can be readily attacked by trypsin. Single-stranded deoxy- and ribopolynucleotides have the same effect as double-stranded DNA's, with the exception of poly d(A)(,n), poly d(I)(,n) and poly r(C)(,n) which do not bind or confer a conformation on CRP which is poorly attacked by trypsin. The 10,000 dalton fragment produced by trypsin digestion of the CRP-DNA complex has an N-terminal sequence identical to that of native CRP and terminates at Lys 89. The 6,000 fragment extends from Val 131 up to Arg 185.;Modification of the arginines with phenylglyoxal or butanedione results in loss of DNA binding activity. cAMP-CRP incorporates more ('14)C-phenylglyoxal than unliganded CRP. Titration of the arginines with ('14)C-phenylglyoxal to where over 90% of the DNA binding activity is lost results in incorporation of one mole of reagent per mole of subunit. Kinetic analysis suggests that incorporation of one molecule of butanedione per molecule of CRP is sufficient to inactivate ('3)H-d(A-T)(,n) binding activity. Modification by phenylglyoxal or butanedione does not affect cAMP binding activity. CRP modified with butanedione is rendered sensitive to chymotryptic attack in the absence of cAMP.
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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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Biochemistry
