Browsing by Subject "Cyclic adenylic acid"
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Item cAMP-mediated effects on CRP subunit interactions(Texas Tech University, 2001-08) Tomlinson, Steven RichardCyclic 3':5'-adenosine monophosphate (cAMP) receptor protein (CRP) of Escherichia coli is a dimeric 47 kDa protein composed of identical subunits. Each subunit can bind cyclic nucleotide. CRP exhibits negative cooperativity in binding cAMP and the current model supports three different CRP conformers: apo-CRP the CRP:(cAMP)^1 complex, and the CRP: cAMP)^2 complex. In the presence of cAMP. CRP binds to specific DNA sequences located upstream of a number of E. coli promoters. The CRP:(c.AMP)^1 complex regulates transcription from these promoters. The conformational changes that activate CRP are not completely understood. Weber and Steitz (J. Mol.Biol. 1987) predicted that upon binding cAMP. CRP subunits are likeh to reorient. To test this hypothesis amino acids along the CRP C helix were substituted with cysteine to allow intermolecular disulfide bond formation. Disulfide crosslinked CRP's were analyzed to confirm the presence of intermolecular positional pair disulfide bonds. The distribution of these disulfide bonds was analyzed over a wide range of cAMP concentrations. These experiments show that while there were only modest cAMPdependent changes in disulfide bond distribution, the C-proximal end of the C helix exhibited a surprising degree of flexibility. During the course of this study, leucine (L) at position 124, located in the cyclic nucleotide binding pocket of CRP, was shown to be important in CRP activation. The substitution of L124 with alanine (A) decreased the affinity of CRP for the first molecule of cAMP by about one order of magnitude and decreased negative cooperativity in cAMP binding by 55-fold. In the presence of cAMP cells containing L124A CRP did not induce B-galactosidase in vivo. In vitro studies showed cAMP binding to L124A failed to produce conformational changes in the CRP. The hydrophobic side chain bulk of L124, which packs against cAMP, appears important in cAMP binding and in CRP activation. Finally, an assay for monitoring CRP subunit exchange was developed to assess the affect of amino acid substitutions at CRP position 99. The results of this study provide conclusive evidence that tyrosine 99 plays an important role in stabilizing the association of CRP subunits.Item DNA structure and cyclic AMP receptor protein-mediated lactose operon promoter activation(Texas Tech University, 1992-12) Gang, Jong-backThe cyclic adenosine 3';5' monophosphate (cAMP) receptor protein (CRP) complexed with cAMP binds a site upsu-eam of the lactose operon promoter (lacP) and activates transcription of the operon. The binding of the wild-type and two mutant forms of CRP to lacF DNA has been characterized with respect to the CRP-induced DNA bending angle and the CRP bending locus. DNase I footprint experiments were conducted to develop a better understanding of the requirements for the interaction of each of these three forms of CRP and lacP DNA. This study suggested that CRP-mediated transcription activation of lacP occurs through a mechanism that includes a role for protein-protein interaction between CRP and RNA polymerase. An intramolecular DNA ligation assay was utilized to determine the DNA molar cyclization factor when bound by each of these three forms of CRP. Six DNA fragments, each containing the CRP binding site and a short phasing sequence, were constructed and served as substrates for this assay. Ligation assays were done in the absence of CRP, in the presence of either wild-type or mutant CRP, or in the presence of the wild-type or mutant CRP and cyclic nucleotide.Item DNA superhelical density and CRP-mediated lac promoter activation(Texas Tech University, 1991-05) Lee, Hae KyungThe cyclic AMP receptor protein (CRP) functions in Escherichia coli in the regulation of several catabolite-sensitive operons. Cyclic AMP (cAMP) forms a complex with CRP which binds to specific DNA sites near the promoter of lactose operon {lacP) and activates the rate of its transcription by RNA polymerase. Mutant forms of the proteins (CRP*) that function independentiy of cAMP have been described. Studies were conducted to assess the response of the wild-type and mutant forms of CRP to changes in template DNA structure. To investigate the effect of varying template superhelical density (SHD) on CRP and CRP*-mediated lacP activity, in vitro transcription assays were performed. A lacP containing plasmid DNA, pJB3.5d/ac, was constructed and a series of topoisomers ranging from SHD = 0.000 to -0.098 were generated. The results of both full length transcripts and abortive initiation assays showed that CRP-mediated lacP activity was low at low template SHD and increased with increased template SHD under all conditions tested. Each mutant form of the protein was, however, unique in the range of DNA SHD providing maximal lacP stimulation. In addition, two mutant forms of CRP promoted RNA polymerase:/acP open complex formation in the absence of cAMP at half-times comparable to the CRPicAMP complex. The open complex formation half-times in reactions that contained wild-type CRPicAMP complex decreased with increasing SHD and are in good agreement with the published data. DNA:CRP binding properties of topoisomers were determined in experiments that utilize a product of in vivo recombination product, a small (451 base pairs) covalently closed circular DNA containing lacP region. The gel mobility shift assay on DNA:CRP:cAMP complex showed that in all DNA SHD examined, both wild-type and the mutant forms of CRP cause a retardation in migration rate, while in the absence of cAMP no difference in migration was observed. From the abortive initiation assay on 451 base pair (bp) DNA, it was found that lacP contained on the 451 bp circle does not show the same degree of cAMP dependence as lacP contained on a larger covalendy closed circle.Item Intersubunit interaction changes resulting from cAMP activation of CRP(Texas Tech University, 1999-05) Riek, Bradley CDimeric cyclic adenosine 3',5' monophosphate receptor protein (CRP) complexes with cAMP to form an asymmetric CRP:(cAMP)1 complex. CRP:(cAMP)1 binds upstream of lacP to activate the promoter via interactions with RNA polymerase. The goal of this work was to determine whether the cAMP-bound subunit of CRP:(c AMP)I must be positioned exclusively in one orientation with respect to RNA polymerase to activate transcription. In vitro transcription experiments were designed to determine whether the cAMP-bound subunit must be oriented in either the promoter-proximal or the promoter-distal position to activate transcription. Site-directed mutagenesis was used to construct a CRP species that had altered affinity for DNA binding and decreased affinity for cAMP. Incubation of this mutant with WT CRP produced a heterodimeric species containing one WT subunit and one mutant subunit. At low concentrations of cAMP, only the WT subunit binds cAMP, producing CRP:(cAMP)i. The differing subunit affinities for specific DNA sequences allowed for orientation of CRP:(cAMP)i on two asymmetric templates. Mutagenesis of the CRP consensus binding sequence produced, via point mutations in either CRPbinding half-site, two templates capable of orienting the cAMP-bound subunit in either the promoter-proximal or promoter-distal position. In vitro transcription assays showed that the heterodimer activated transcription with the cAMP-bound subunit in either orientation. This data indicates the CRP:(cAMP)1 complex is not required to orient its cAMP-bound subunit with respect to RNA polymerase to activate lacP.Item Mutagenesis of cyclic AMP receptor protein: targeting positions 72 and 82 of the cyclic nucleotide binding pocket(Texas Tech University, 1992-12) Belduz, Ali AsmanThe cyclic adenosine 3',5' monophosphate (cAMP): cAMP receptor protein (CRP) complex functions to promote transcription activation of several operons in Escherichia coli. The complex binds to specific DNA sites located upstream of, for example, the lactose operon promoter (lacP). This activates lacP and allows high level expression of lactose operon structural gene sequences. Five specific amino acid residues are predicted to play a role in the interaction of cAMP with CRP. The purpose of this work was to test the role of two amino acid residues, glutamate at position 72 and arginine at position 82, in facilitating both cAMP binding and cAMP-mediated activation of CRP. Mutants at positions 072 and 082 in the cyclic nucleotide binding pocket of the CRP were constructed by site directed mutagenesis. Glutamate at position 072 was substituted by either leucine, glutamine, or aspartate. Arginine at the position 082 was substituted by either lysine, histidine, leucine, isoleucine, or glutamine. A parallel set of substitutions was constructed in a CRP*, or cAMP-independent, form of the protein to determine the effects of the amino acid substitutions on CRP conformation. Each mutant form of CRP was characterized in vivo with respect to sugar utilization, and through quantitative pgalactosidase activity and in vitro by measurement of cAMP binding activity under near physiological salt concentrations. The results of this study confirm a role for both glutamate at position 72 and arginine at position 82 in cAMP binding and activation of CRP; neither aspartate at position 72 nor lysine at position 82 could effectively substitute for glutamate or arginine in an otherwise wild-type CRP. The results of this study also indicate that cGMP, a competitive inhibitor of cAMP binding to CRP, binds the cyclic nucleotide binding pocket differendy than cAMP.