CHEMICAL MODIFICATION OF THE SIGMA SUBUNIT OF ESCHERICHIA COLI DNA-DEPENDENT RIBONUCLEIC ACID POLYMERASE (RNA).
Item
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Title
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CHEMICAL MODIFICATION OF THE SIGMA SUBUNIT OF ESCHERICHIA COLI DNA-DEPENDENT RIBONUCLEIC ACID POLYMERASE (RNA).
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Identifier
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AAI8401949
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identifier
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8401949
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Creator
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NARAYANAN, CHITTAMPALLI SHESHA CHAR.
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Contributor
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Prof. Joseph S. Krakow
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Date
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1983
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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, General
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Abstract
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The function of lysine, arginine, cysteine and carboxylic amino acid (glutamic and aspartic) residues of sigma was studied using chemical modification by group specific reagents. Following modification of 3 arginines with phenylglyoxal or 3 cysteines with N-ethylmaleimide (NEM) or 5 lysines with trinitrobenzene sulfonate sigma activity was lost. Analysis of the kinetic data for inactivation indicated that one lysine or arginine or cysteine residue with estimated pK values of 9, 8 and 8 respectively is essential for sigma activity. At low NEM concentration alkylation was limited to a nonessential cysteine. Modification of lysine or arginine or cysteine residues had no observable effect on the binding affinity of inactivated sigma to the core polymerase but inhibited promoter recognition and initiation effects of sigma on core polymerase. Holoenzyme containing the modified sigma was also able to bind DNA. Modification of aspartic and/or glutamic acid residues with the water soluble carbodiimides, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) or 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMC) resulted in loss of sigma activity. The inactivation and kinetic data indicated that one carboxylic amino acid is essential for sigma activity. Sigma modified with EDC, CMC or EDC in the presence of glycine was inactive in supporting promoter binding and inactivation by core polymerase. Reaction with EDC plus (('3)H)glycine resulted in the incorporation of glycine into sigma. The (('3)H)glycine-sigma is unable to form a stable holoenzyme complex. Limited proteolytic digestion of modified sigma indicated a change in the conformation of sigma following the modification.;Autoradiography of the cyanogen bromide fragments of radiolabelled sigma suggested that the critical lysyl or cysteinyl groups are not in the first 288 amino acids from the N-terminus. The nonessential cysteinyl group was identified as cys132. The critical carboxyl group is in the first 288 amino acids from the N-terminus. It is possible that the N-terminal half of sigma is involved in the interaction of sigma with the core polymerase and the C-terminal half of sigma containing the critical lysyl and cysteinyl groups is involved in the catalytic function of sigma.
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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
