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AraC Protein

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Robert Schleif – One of the best experts on this subject based on the ideXlab platform.

  • Helical Behavior of the Interdomain Linker of the Escherichia coli AraC Protein.
    Biochemistry, 2019
    Co-Authors: Matthew Brown, Robert Schleif

    Abstract:

    In Escherichia coli, the dimeric AraC Protein actively represses transcription from the l-arabinose araBAD operon in the absence of arabinose but induces transcription in its presence. Here we provide evidence that, in shifting from the repressing to the inducing state, the behavior of the interdomain linker shifts from that of an α helix to that of a more flexible form. In vivo and in vitro experiments show that AraC with a linker sequence that favors helix formation is shifted toward the repressing state in the absence and presence of arabinose. Conversely, AraC containing a linker sequence that is unfavorable for helix formation is shifted toward the inducing state. Experiments in which the presumed helical linker is shortened or lengthened, Protein helical twist experiments, are also consistent with a helix transition mechanism. Previous experiments have shown that, upon the binding of arabinose, the apparent rigidity with which the DNA binding domains of AraC are held in space decreases. Thus, arabin…

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  • Arabinose Alters Both Local and Distal H–D Exchange Rates in the Escherichia coli AraC Transcriptional Regulator
    Biochemistry, 2019
    Co-Authors: Alexander Tischer, Robert Schleif, Matthew Brown, Matthew Auton

    Abstract:

    In the absence of arabinose, the dimeric Escherichia coli regulatory Protein of the l-arabinose operon, AraC, represses expression by looping the DNA between distant half-sites. Binding of arabinose to the dimerization domains forces AraC to preferentially bind two adjacent DNA half-sites, which stimulates RNA polymerase transcription of the araBAD catabolism genes. Prior genetic and biochemical studies hypothesized that arabinose allosterically induces a helix-coil transition of a linker between the dimerization and DNA binding domains that switches the AraC conformation to an inducing state [Brown, M. J., and Schleif, R. F. (2019) Biochemistry, preceding paper in this issue (DOI: 10.1021/acs.biochem.9b00234)]. To test this hypothesis, hydrogen-deuterium exchange mass spectrometry was utilized to identify structural regions involved in the conformational activation of AraC by arabinose. Comparison of the hydrogen-deuterium exchange kinetics of individual dimeric dimerization domains and the full-length dimeric AraC Protein in the presence and absence of arabinose reveals a prominent arabinose-induced destabilization of the amide hydrogen-bonded structure of linker residues (I167 and N168). This destabilization is demonstrated to result from an increased probability to form a helix capping motif at the C-terminal end of the dimerizing α-helix of the dimerization domain that preceeds the interdomain linker. These conformational changes could allow for quaternary repositioning of the DNA binding domains required for induction of the araBAD promoter through rotation of peptide backbone dihedral angles of just a couple of residues. Subtle changes in exchange rates are also visible around the arabinose binding pocket and in the DNA binding domain.

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  • A genetic and physical study of the interdomain linker of E. Coli AraC Protein–a trans-subunit communication pathway.
    Proteins: Structure Function and Bioinformatics, 2016
    Co-Authors: Fabiana Malaga, Michael E. Rodgers, Ory Mayberry, David J. Park, Dmitri Toptygin, Robert Schleif

    Abstract:

    Genetic experiments with full length AraC and biophysical experiments with its dimerization domain plus linker suggest that arabinose binding to the dimerization domain changes the properties of the inter-domain linker which connects the dimerization domain to the DNA binding domain via interactions that do not depend on the DNA binding domain. Normal AraC function was found to tolerate considerable linker sequence alteration excepting proline substitutions. The proline substitutions partially activate transcription even in the absence of arabinose and hint that a structural shift between helix and coil may be involved. To permit fluorescence anisotropy measurements that could detect arabinose-dependent dynamic differences in the linkers, IAEDANS was conjugated to a cysteine residue substituted at the end of the linker of dimerization domain. Arabinose, but not other sugars, decreased the steady-state anisotropy, indicating either an increase in mobility and/or an increase in the fluorescence lifetime of the IAEDANS. Time-resolved fluorescence measurements showed that the arabinose-induced anisotropy decrease did not result from an increase in the excited-state lifetime. Hence arabinose-induced decreases in anisotropy appear to result from increased tumbling of the fluorophore. Arabinose did not decrease the anisotropy in mutants incapable of binding arabinose nor did it alter the anisotropy when IAEDANS was conjugated elsewhere in the dimerization domain. Experiments with heterodimers of the dimerization domain showed that the binding of arabinose to one subunit of the dimer decreases the fluorescence anisotropy of only a fluorophore on the linker of the other subunit.

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Gary Wilcox – One of the best experts on this subject based on the ideXlab platform.

  • Mutations in the AraC gene of Salmonella typhimurium LT2 which affect both activator and auto-regulatory functions of the AraC Protein
    Gene, 1992
    Co-Authors: Patrick Clarke, Jar-how Lee, Kathleen A. Burke, Gary Wilcox

    Abstract:

    Abstract The AraC gene encodes a regulatory Protein, AraC, that acts as both an activator and a repressor of transcription of the genes involved in the transport and catabolism of l -arabinose in Salmonella typhimurium LT2. Five AraC mutants which have altered regulatory properties were chAraCterized. All are point mutations which would result in amino acid substitutions near the C terminus of AraC. Each mutation results in altered activator and auto-regulatory AraC function in vivo. In vitro DNA-binding assays showed that three mutant AraC have measurable lowered affinity for aca controlling site DNA. The data are consistent with a model in which there is a DNA-binding domain in the C terminus of AraC which functions in both activation and repression.

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Sanjib Bhakta – One of the best experts on this subject based on the ideXlab platform.

  • ChAraCterisation of a putative AraC transcriptional regulator from Mycobacterium smegmatis
    Tuberculosis, 2014
    Co-Authors: Dimitrios Evangelopoulos, Antima Gupta, Nathan A. Lack, Arundhati Maitra, Annemieke Ten Bokum, Sharon L. Kendall, Edith Sim, Sanjib Bhakta

    Abstract:

    MSMEG_0307 is annotated as a transcriptional regulator belonging to the AraC Protein family and is located adjacent to the arylamine N-acetyltransferase (nat) gene in Mycobacterium smegmatis, in a gene cluster, conserved in most environmental mycobacterial species. In order to elucidate the function of the AraC Protein from the nat operon in M. smegmatis, two conserved palindromic DNA motifs were identified using bioinformatics and tested for Protein binding using electrophoretic mobility shift assays with a recombinant form of the AraC Protein. We identified the formation of a DNA:AraC Protein complex with one of the motifs as well as the presence of this motif in 20 loci across the whole genome of M. smegmatis, supporting the existence of an AraC controlled regulon. To chAraCterise the effects of AraC in the regulation of the nat operon genes, as well as to gain further insight into its function, we generated a ΔAraC mutant strain where the AraC gene was replaced by a hygromycin resistance marker. The level of expression of the nat and MSMEG_0308 genes was down-regulated in the ΔAraC strain when compared to the wild type strain indicating an activator effect of the AraC Protein on the expression of the nat operon genes.

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  • NON-TUBERCULOUS MYCOBACTERIA: GENERAL ChAraCterisation of a putative AraC transcriptional regulator from Mycobacterium smegmatis
    , 2014
    Co-Authors: Dimitrios Evangelopoulos, Antima Gupta, Nathan A. Lack, Arundhati Maitra, Sharon L. Kendall, Edith Sim, Sanjib Bhakta

    Abstract:

    summary MSMEG_0307 is annotated as a transcriptional regulator belonging to the AraC Protein family and is located adjacent to the arylamine N-acetyltransferase (nat) gene in Mycobacterium smegmatis, in a gene cluster, conserved in most environmental mycobacterial species. In order to elucidate the function of the AraC Protein from the nat operon in M. smegmatis, two conserved palindromic DNA motifs were identified using bioinformatics and tested for Protein binding using electrophoretic mobility shift assays with a recombinant form of the AraC Protein. We identified the formation of a DNA:AraC Protein complex with one of the motifs as well as the presence of this motif in 20 loci across the whole genome of M. smegmatis, supporting the existence of an AraC controlled regulon. To chAraCterise the effects of AraC in the regulation of the nat operon genes, as well as to gain further insight into its function, we generated a DAraC mutant strain where the AraC gene was replaced by a hygromycin resistance marker. The level of expression of the nat and MSMEG_0308 genes was down-regulated in the DAraC strain when compared to the wild type strain indicating an activator effect of the AraC Protein on the expression of the nat operon genes. © 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license

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