Active Role of the Interdomain Linker of AraC

Seedorff, Jennifer E.; Schleif, Robert

doi:10.1128/jb.05339-11

Cited by 20 publications

(23 citation statements)

References 40 publications

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“…The results are striking in that nearly any substitution lacking proline behaves as wild type, and nearly any substitution in the linker containing proline is impaired in its repression abilities. Because of this and an earlier finding that introduction of a proline residue into the linker region resulted in impaired repression, we also explicitly generated proline substitutions in each of the linker residues 168–172, Table . Repression in each of these was also impaired.…”

Section: Resultsmentioning

confidence: 99%

“…In light of the failures to detect an arm‐DBD interaction we have extended consideration to the possibility that the binding of arabinose, either directly via the N‐terminal arms or indirectly, affects the inter‐domain linker, residues 168–174, that connects each dimerization domain and a DNA binding domain. Although initial genetics experiments suggested that the repressing and inducing activities of AraC are not sensitive to sequence alterations in the linker, more recent and more precisely targeted mutational and biophysical studies have indicated that some mutations in the linker can reduce the protein's ability to repress p BAD . This article describes an expanded genetic analysis of linker mutations and fluorescence anisotropy measurements that show that binding of arabinose to the dimerization domain alters the motion of a conjugated fluorophore.…”

Section: Introductionmentioning

confidence: 99%

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A genetic and physical study of the interdomain linker ofE. ColiAraC protein-atrans-subunit communication pathway

et al. 2016

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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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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A genetic and physical study of the interdomain linker ofE. ColiAraC protein-atrans-subunit communication pathway

et al. 2016

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“…Protein members of the AraC family have two structural domains: the C-terminal DNA binding domain and the N-terminal signaling domain, connected by a relatively unstructured linker (Mahon et al, 2010;Seedorff and Schleif, 2011). The DNA binding domain comprises a ca.…”

Section: Introductionmentioning

confidence: 99%

A large family of anti‐activators accompanying XylS/AraC family regulatory proteins

Santiago

Yan

Tran

et al. 2016

Molecular Microbiology

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Summary AraC Negative Regulators (ANR) suppress virulence genes by directly down-regulating AraC/XylS members in Gram-negative bacteria. In this study we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays, and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR-activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC-like member AggR. ANR-AggR binding disrupted AggR dimerization and prevented AggR-DNA binding. ANR homologs of Vibrio cholera, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α-helices. Site-directed mutagenesis studies suggest that at least predicted α-helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners.

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“…AraC is composed of an N-terminal arabinose-binding and dimerization domain and a C-terminal DNA-binding domain. The expression of the araBAD operon and the AraC-encoding gene (araC), which are divergently transcribed, is repressed by binding of AraC to the promoter region forming a DNA loop in the absence of L-arabinose (13)(14)(15). In the presence of L-arabinose, three promoters, ParaBAD, ParaE, and ParaFGH, are activated, and ParaC is derepressed (16).…”

mentioning

confidence: 99%

The LacI-Type Transcriptional Regulator AraR Acts as an l -Arabinose-Responsive Repressor of l -Arabinose Utilization Genes in Corynebacterium glutamicum ATCC 31831

et al. 2014

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bThe Corynebacterium glutamicum ATCC 31831 araBDA operon consists of three L-arabinose catabolic genes, upstream of which the galM, araR, and araE genes are located in opposite orientation. araR encodes a LacI-type transcriptional regulator that negatively regulates the L-arabinose-inducible expression of araBDA and araE (encoding an L-arabinose transporter), through a mechanism that has yet to be identified. Here we show that the AraR protein binds in vitro to three sites: one upstream of araBDA and two upstream of araE. We verify that a 16-bp consensus palindromic sequence is essential for binding of AraR, using a series of mutations introduced upstream of araB in electrophoretic mobility shift assays. Moreover, the DNA-binding activity of AraR is reduced by L-arabinose. We employ quantitative reverse transcription-PCR (qRT-PCR) analyses using various mutant strains deficient in L-arabinose utilization genes to demonstrate that the prominent upregulation of araBDA and araE within 5 min of L-arabinose supplementation is dependent on the uptake but independent of the catabolism of L-arabinose. Similar expression patterns, together with the upregulation by araR disruption without L-arabinose, are evident with the apparent galM-araR operon, although attendant changes in expression levels are much smaller than those realized with the expression of araBDA and araE. The AraR-binding site upstream of araB overlaps the ؊10 region of the divergent galM promoter. These observations indicate that AraR acts as a transcriptional repressor of araBDA, araE, and galM-araR and that L-arabinose acts as an intracellular negative effector of the AraR-dependent regulation.

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Active Role of the Interdomain Linker of AraC

Cited by 20 publications

References 40 publications

A genetic and physical study of the interdomain linker ofE. ColiAraC protein-atrans-subunit communication pathway

A genetic and physical study of the interdomain linker ofE. ColiAraC protein-atrans-subunit communication pathway

A large family of anti‐activators accompanying XylS/AraC family regulatory proteins

The LacI-Type Transcriptional Regulator AraR Acts as an l -Arabinose-Responsive Repressor of l -Arabinose Utilization Genes in Corynebacterium glutamicum ATCC 31831

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