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Robert Schleif - One of the best experts on this subject based on the ideXlab platform.
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Arabinose Alters Both Local and Distal H–D Exchange Rates in the Escherichia coli AraC Transcriptional Regulator
Biochemistry, 2019Co-Authors: Alexander Tischer, Robert Schleif, Matthew Brown, Matthew AutonAbstract: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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Helical Behavior of the Interdomain Linker of the Escherichia coli AraC Protein.
Biochemistry, 2019Co-Authors: Matthew Brown, Robert SchleifAbstract: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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A genetic and physical study of the interdomain linker of E. Coli AraC Protein--a trans-subunit communication pathway.
Proteins: Structure Function and Bioinformatics, 2016Co-Authors: Fabiana Malaga, Michael E. Rodgers, Ory Mayberry, David J. Park, Dmitri Toptygin, Robert SchleifAbstract: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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Understanding the basis of a class of paradoxical mutations in AraC through simulations
Proteins: Structure Function and Bioinformatics, 2012Co-Authors: Ana Damjanović, Benjamin T. Miller, Robert SchleifAbstract:Most mutations at position 15 in the N-terminal arm of the regulatory Protein AraC leave the Protein incapable of responding to arabinose and inducing the Proteins required for arabinose catabolism. Mutations at other positions of the arm do not have this behavior. Simple energetic analysis of the interactions between the arm and bound arabinose do not explain the uninducibility of AraC with mutations at position 15. Extensive molecular dynamics (MD) simulations, carried out largely on the Open Science Grid, were done of the wild-type Protein with and without bound arabinose and of all possible mutations at position 15, many of which were constructed and measured for this work. Good correlation was found for deviation of arm position during the simulations and inducibility as measured in vivo of the same mutant Proteins. Analysis of the MD trajectories revealed that preservation of the shape of the arm is critical to inducibility. To maintain the correct shape of the arm, the strengths of three interactions observed to be strong in simulations of the wild-type AraC Protein need to be preserved. These interactions are between arabinose and residue 15, arabinose and residues 8-9, and residue 13 and residue 15. The latter interaction is notable because residues L9, Y13, F15, W95, and Y97 form a hydrophobic cluster which needs to be preserved for retention of the correct shape.
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Heterodimers Reveal That Two Arabinose Molecules Are Required for the Normal Arabinose Response of AraC
Biochemistry, 2012Co-Authors: Michael E. Rodgers, Robert SchleifAbstract:AraC Protein, which regulates expression of the L- arabinose operon in Escherichia coli, is a dimer whose DNA binding affinity for pairs of DNA half-sites is controlled by arabinose. Here we have addressed the question of whether the arabinose response of AraC requires the binding of one or two molecules of arabinose. This was accomplished by measuring the DNA dissociation rates of wild-type AraC and heterodimeric AraC constructs in which one subunit is capable of binding arabinose and the other subunit does not bind arabinose. Solutions consisting entirely of heterodimers were formed by spontaneous subunit exchange between two different homodimers, with heterodimers being trapped by the formation of an intersubunit disulfide bond between cysteine residues strategically positioned within the dimerization interface. We found that the normal arabinose response of AraC requires the binding of two arabinose molecules. These results provide additional constraints on mechanistic models for the action of AraC.
Gary Wilcox - One of the best experts on this subject based on the ideXlab platform.
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Mutations in the AraC gene of Salmonella typhimurium LT2 which affect both activator and auto-regulatory functions of the AraC Protein
Gene, 1992Co-Authors: Patrick Clarke, Kathleen A. Burke, Jar-how Lee, Gary WilcoxAbstract: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.
Sanjib Bhakta - One of the best experts on this subject based on the ideXlab platform.
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ChAraCterisation of a putative AraC transcriptional regulator from Mycobacterium smegmatis
Tuberculosis, 2014Co-Authors: Dimitrios Evangelopoulos, Antima Gupta, Nathan A. Lack, Arundhati Maitra, Annemieke Ten Bokum, Sharon L. Kendall, Edith Sim, Sanjib BhaktaAbstract: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
2014Co-Authors: Dimitrios Evangelopoulos, Antima Gupta, Nathan A. Lack, Arundhati Maitra, Sharon L. Kendall, Edith Sim, Sanjib BhaktaAbstract: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
Silvia A. Bustos - One of the best experts on this subject based on the ideXlab platform.
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Reaching out. Locating and lengthening the interdomain linker in AraC Protein.
Journal of Molecular Biology, 1994Co-Authors: Rebecca J. Eustance, Silvia A. Bustos, Robert SchleifAbstract:A genetic method was developed to determine, in Proteins, areas which are tolerant of insertions and deletions. Attractive candidates for these areas are linker regions. Such a region was found to include positions 171 to 178 in the Escherichia coli regulatory Protein AraC. Independent biochemical methods identified amino acid residues 11 to 170 as the minimal dimerization domain of AraC, and amino acid residues 178 to 286 out of the 291 residue Protein as the minimal DNA-binding domain. Hence, by both the genetic and biochemical approaches, the interdomain linking region was determined to include amino acid residues 171 to 177. The properties of altered Proteins were examined using templates with AraC half-sites more widely separated than in the wild-type case. Both AraC Protein containing an insertion in the interdomain linker region and a Protein consisting of the minimal functional dimerization and DNA-binding domains separated by a 39 amino acid residue linker were able to bind to and function on such a DNA site. In vitro, the Proteins with longer linkers bound substantially more stably than wild-type AraC to the DNA containing half-sites for AraC separated by an extra two helical turns of DNA. In vivo on an ara promoter with the more widely separated AraC half-sites, the Proteins could activate transcription much better than wild-type AraC.
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Functional domains of the AraC Protein.
Proceedings of the National Academy of Sciences, 1993Co-Authors: Silvia A. Bustos, Robert SchleifAbstract:The AraC Protein, which regulates the L-arabinose operons in Escherichia coli, was dissected into two domains that function in chimeric Proteins. One provides a dimerization capability and binds the ligand arabinose, and the other provides a site-specific DNA-binding capability and activates transcription. In vivo and in vitro experiments showed that a fusion Protein consisting of the N-terminal half of the AraC Protein and the DNA-binding domain of the LexA repressor dimerizes, binds well to a LexA operator, and represses expression of a LexA operator-beta-galactosidase fusion gene in an arabinose-responsive manner. In vivo and in vitro experiments also showed that a fusion Protein consisting of the C-terminal half of the AraC Protein and the leucine zipper dimerization domain from the C/EBP transcriptional activator binds to araI and activates transcription from a PBAD promoter-beta-galactosidase fusion gene. Dimerization was necessary for occupancy and activation of the wild-type AraC binding site.
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Functional domains oftheAraCProtein
1993Co-Authors: Silvia A. Bustos, Robertf . SchleifAbstract:ABSTRACT The AraC Protein, which regulates the L-arabinoseoperons in Escherichiacoi, wasdissectedintotwo domains that function in chimeric Proteins. One provides a dimerization capability andbindsthe ligandarabinose, andthe other provides a site-specific DNA-binding capability and activatestanscription. In vivoand in vitroexperiments showed that a fusion Protein consisting ofthe N-terminal halfofthe AraC Protein and the DNA-binding domain of the LexA repressor dimerizes, binds weDl to a LexA operator, and repressesexpressionofaLexA operator-3-galactosidase fusiongene in an arabinose-responsive manner. In vivo andin vitroexperiments alsoshowedthatafusion Proteinconsistingof the C-terminal half of the AraC Protein and the leucine zipperdimerization domain from the C/EBPtranscriptional activa- tor binds to aral and activates transcription from a PB.4D promoter-,-galactosidase fusion gene. Dimerization wasnec- essary for occupancy and activation of the wild-type AraCbinding site. MostoftheProteinswhose
Patrick Clarke - One of the best experts on this subject based on the ideXlab platform.
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Mutations in the AraC gene of Salmonella typhimurium LT2 which affect both activator and auto-regulatory functions of the AraC Protein
Gene, 1992Co-Authors: Patrick Clarke, Kathleen A. Burke, Jar-how Lee, Gary WilcoxAbstract: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.