Strengthened Arm-Dimerization Domain Interactions in AraC

Wu, Martin; Schleif, Robert

doi:10.1074/jbc.m008705200

Cited by 21 publications

(24 citation statements)

References 20 publications

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“…The light switch model proposes that, in the absence of arabinose, the interaction of the AraC N-terminal arm with the C-terminal DNA-binding domain constrains the subunits of the AraC dimer in an orientation that makes it energetically favorable to bind to distal (O2 and I1) rather than adjacent (I1 and I2) targets (11,25). This model is supported by genetic analyses, notably, mutations that alter amino acids in the AraC N-terminal arm that result in AraC-dependent activation of paraBAD in the absence of arabinose (19,21,22,34,35).…”

Section: Discussionsupporting

confidence: 49%

Mutational Analysis of the Escherichia coli melR Gene Suggests a Two-State Concerted Model To Explain Transcriptional Activation and Repression in the Melibiose Operon

et al. 2006

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Transcription of the Escherichia coli melAB operon is regulated by the MelR protein, an AraC family member whose activity is modulated by the binding of melibiose. In the absence of melibiose, MelR is unable to activate the melAB promoter but autoregulates its own expression by repressing the melR promoter. Melibiose triggers MelR-dependent activation of the melAB promoter and relieves MelR-dependent repression of the melR promoter. Twenty-nine single amino acid substitutions in MelR that result in partial melibiose-independent activation of the melAB promoter have been identified. Combinations of different substitutions result in almost complete melibiose-independent activation of the melAB promoter. MelR carrying each of the single substitutions is less able to repress the melR promoter, while MelR carrying some combinations of substitutions is completely unable to repress the melR promoter. These results argue that different conformational states of MelR are responsible for activation of the melAB promoter and repression of the melR promoter. Supporting evidence for this is provided by the isolation of substitutions in MelR that block melibiose-dependent activation of the melAB promoter while not changing melibiose-independent repression of the melR promoter. Additional experiments with a bacterial two-hybrid system suggest that interactions between MelR subunits differ according to the two conformational states.

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Section: Discussionsupporting

confidence: 49%

Mutational Analysis of the Escherichia coli melR Gene Suggests a Two-State Concerted Model To Explain Transcriptional Activation and Repression in the Melibiose Operon

et al. 2006

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“…The finding that RhaS-CTD activates transcription very well in the absence of RhaS-NTD suggests that there may be inhibition of full-length RhaS activity in the absence of ligand. The light-switch mechanism used by AraC to respond to its ligand arabinose also involves inhibition in the absence of ligand (14,27,29,31,45,46). However, our more recent results suggest that the L-rhamnose response of RhaS most likely involves an active stimulation of activity in the presence of L-rhamnose (Kolin and Egan, unpublished [see below]).…”

Section: Discussionmentioning

confidence: 79%

Transcription Activation by the DNA-Binding Domain of the AraC Family Protein RhaS in the Absence of Its Effector-Binding Domain

et al. 2007

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The Escherichia coli L-rhamnose-responsive transcription activators RhaS and RhaR both consist of two domains, a C-terminal DNA-binding domain and an N-terminal dimerization domain. Both function as dimers and only activate transcription in the presence of L-rhamnose. Here, we examined the ability of the DNAbinding domains of RhaS (RhaS-CTD) and RhaR (RhaR-CTD) to bind to DNA and activate transcription. RhaS-CTD and RhaR-CTD were both shown by DNase I footprinting to be capable of binding specifically to the appropriate DNA sites. In vivo as well as in vitro transcription assays showed that RhaS-CTD could activate transcription to high levels, whereas RhaR-CTD was capable of only very low levels of transcription activation. As expected, RhaS-CTD did not require the presence of L-rhamnose to activate transcription. The upstream half-site at rhaBAD and the downstream half-site at rhaT were found to be the strongest of the known RhaS half-sites, and a new putative RhaS half-site with comparable strength to known sites was identified. Given that cyclic AMP receptor protein (CRP), the second activator required for full rhaBAD expression, cannot activate rhaBAD expression in a ⌬rhaS strain, it was of interest to test whether CRP could activate transcription in combination with RhaS-CTD. We found that RhaS-CTD allowed significant activation by CRP, both in vivo and in vitro, although full-length RhaS allowed somewhat greater CRP activation. We conclude that RhaS-CTD contains all of the determinants necessary for transcription activation by RhaS.The RhaS protein functions to activate transcription of two of the operons in the Escherichia coli L-rhamnose regulon in response to the availability of L-rhamnose (11,40). The two operons rhaBAD and rhaT encode the L-rhamnose catabolic enzymes (L-rhamnulokinase, L-rhamnose isomerase, and Lrhamnulose-1-phosphate aldolase) (2, 25) and an L-rhamnoseproton symporter that is responsible for transporting L-rhamnose into the cell (35), respectively. RhaS is encoded in an operon that also encodes a second L-rhamnose-responsive transcription activator, RhaR (37). RhaR activates transcription of the operon that encodes the two activator proteins, rhaSR (37, 38). All three operons in the L-rhamnose regulon also require a second activator protein, cyclic AMP (cAMP) receptor protein (CRP), for full transcription activation (11,16,40).

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“…These results are consistent with previous studies indicating substitutions in the N-terminal arm weaken repression in the absence of a ligand. 11,12,20 Understanding the orientation of TAL within the binding pocket will also help to understand molecular recognition and how to better design for selectivity. Notably, molecular docking studies with the current apo-structure of the AraC-TAL1 ligand-binding domain have been inconclusive, in that many potential TAL orientations show similar binding energies and are within the error of the energy calculations in the docking protocol (results not shown).…”

Section: Discussionmentioning

confidence: 99%

Analysis of amino acid substitutions in AraC variants that respond to triacetic acid lactone

et al. 2016

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The Escherichia coli regulatory protein AraC regulates expression of ara genes in response to L-arabinose. In efforts to develop genetically encoded molecular reporters, we previously engineered an AraC variant that responds to the compound triacetic acid lactone (TAL). This variant (named "AraC-TAL1") was isolated by screening a library of AraC variants, in which five amino acid positions in the ligand-binding pocket were simultaneously randomized. Screening was carried out through multiple rounds of alternating positive and negative fluorescence-activated cell sorting. Here we show that changing the screening protocol results in the identification of different TAL-responsive variants (nine new variants). Individual substituted residues within these variants were found to primarily act cooperatively toward the gene expression response. Finally, X-ray diffraction was used to solve the crystal structure of the apo AraC-TAL1 ligand-binding domain. The resolved crystal structure confirms that this variant takes on a structure nearly identical to the apo wild-type AraC ligand-binding domain (root-mean-square deviation 0.93 Å ), suggesting that AraC-TAL1 behaves similar to wild-type with regard to ligand recognition and gene regulation. Our results provide amino acid sequence-function data sets for training and validating AraC modeling studies, and contribute to our understanding of how to design new biosensors based on AraC.

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Strengthened Arm-Dimerization Domain Interactions in AraC

Cited by 21 publications

References 20 publications

Mutational Analysis of the Escherichia coli melR Gene Suggests a Two-State Concerted Model To Explain Transcriptional Activation and Repression in the Melibiose Operon

Mutational Analysis of the Escherichia coli melR Gene Suggests a Two-State Concerted Model To Explain Transcriptional Activation and Repression in the Melibiose Operon

Transcription Activation by the DNA-Binding Domain of the AraC Family Protein RhaS in the Absence of Its Effector-Binding Domain

Analysis of amino acid substitutions in AraC variants that respond to triacetic acid lactone

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