Structure and function of the repressor and operators of the sn-glycerol-3-phosphate regulon of Escherichia coli K-12

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dc.contributor.authorYe, Shanzhangen
dc.contributor.committeechairLarson, Timothy J.en
dc.contributor.committeememberPotts, Malcolmen
dc.contributor.committeememberSitz, Thomas O.en
dc.contributor.committeememberJohnson, John L.en
dc.contributor.committeememberPedersen, Karlen
dc.contributor.departmentBiochemistry and Nutritionen
dc.date.accessioned2014-03-14T21:21:28Zen
dc.date.adate2005-10-19en
dc.date.available2014-03-14T21:21:28Zen
dc.date.issued1990en
dc.date.rdate2005-10-19en
dc.date.sdate2005-10-19en
dc.description.abstractThe glpD gene, which encodes aerobic sn-glycerol 3-phosphate dehydrogenase, and the glpR gene, which encodes a repressor that negatively regulates the expression of the g/p regulon, map near minute 75 on the linkage map of Escherichia coli K-12. In the present study, the nucleotide sequence of the 2895 base pair of DNA containing the glpD control region and the glpE, glpG, glpR genes was determined. The translation initiation codons with adjacent ribosome-binding sites were found for these four genes. The transcription start site of the glpD gene was identified 42 base pairs upstream from the proposed methionine start codon, preceded by a region containing typical -10 and -35 sequences found in bacterial promoters. A binding site for the cyclic AMP-cAMP receptor protein complex was located just upstream from the -35 sequence, centered at position -63. The interaction site for the glp repressor was identified by using DNase I footprinting. This region contained two tandemly repeated sequences which started at the -10 sequence and continued to position +38. The glp repressor contained 252 amino acid residues and had a molecular weight of 28,046 which was deduced from the nucleotide sequence. The position of the initiation codon was verified by determination of the amino acid sequence of the N-terminus of the purified gip repressor. The presumptive helix-turn-helix region of the repressor was located near the N-terminus (amino acids 22 to 41) at a poSition analogous to that found for the operator binding domain of other repressors such as the deo and Jac repressors. The recognition helix of the glp repressor and the nucleotide sequence of the glp operator were very similar to those of the deo system. The presumptive glpR recognition helix was changed to the deoR recognition helix and the sixth amino acid arginine of the recognition helix was changed to alanine by site-directed mutagenesis. The mutant forms of the repressor had a greatly reduced affinity for the glpD operators in vivo, determined by measuring β-galactosidase activity in a strain carrying a glpD-lacZ fusion.en
dc.description.degreePh. D.en
dc.format.extentxiii, 130 leavesen
dc.format.mediumBTDen
dc.format.mimetypeapplication/pdfen
dc.identifier.otheretd-10192005-113257en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-10192005-113257/en
dc.identifier.urihttp://hdl.handle.net/10919/39953en
dc.language.isoenen
dc.publisherVirginia Techen
dc.relation.haspartLD5655.V856_1990.Y4.pdfen
dc.relation.isformatofOCLC# 23734714en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subject.lccLD5655.V856 1990.Y4en
dc.subject.lcshEscherichia coli -- Researchen
dc.titleStructure and function of the repressor and operators of the sn-glycerol-3-phosphate regulon of Escherichia coli K-12en
dc.typeDissertationen
dc.type.dcmitypeTexten
thesis.degree.disciplineBiochemistry and Nutritionen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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