Versatility of the carboxy‐terminal domain of the α subunit of RNA polymerase in transcriptional activation: use of the DNA contact site as a protein contact site for MarA

Dangi, Bindi; Gronenborn, Angela M.; Rosner, Judah L.; Martin, Robert G.

doi:10.1111/j.1365-2958.2004.04250.x

Cited by 47 publications

(62 citation statements)

References 57 publications

(110 reference statements)

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“…This is the case for tcpA, tagA, and acfD. The type 1 interaction is relatively spacing insensitive and may involve an interaction between ␣-CTD and ToxT that is not localized to the DNA, as has been observed with some other AraC family members (4,36). The type 1 interaction is very virstatin sensitive.…”

Section: Discussionmentioning

confidence: 87%

Flexibility of Vibrio cholerae ToxT in Transcription Activation of Genes Having Altered Promoter Spacing

Bellair

Withey

2008

J Bacteriol

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Cholera, a severe diarrheal disease, is caused by ingestion of the gram-negative bacterium Vibrio cholerae. Expression of V. cholerae virulence factors is highly regulated at the transcriptional and posttranscriptional levels by a complex network of proteins and small noncoding RNAs. The direct activator of transcription of most V. cholerae virulence genes is the ToxT protein. ToxT binds to a 13-bp sequence, the toxbox, located upstream of genes in its regulon. However, the organization of toxboxes relative to each other and to the core promoter elements at different genes varies dramatically. At different ToxT-activated genes a single toxbox may be necessary and sufficient for full activation, or pairs of toxboxes organized as either inverted or direct repeats may be required for full activation. Although all toxboxes are located at positions consistent with a class I promoter architecture, the locations of toxboxes relative to the transcription start site also vary from gene to gene. To further assess the ability of ToxT to activate transcription from different configurations relative to the core promoter elements, we constructed promoter-lacZ fusions having altered spacing both between toxbox pairs and between the promoter-proximal toxbox and the ؊35 box at five different ToxT-activated promoters. Our results suggest that that ToxT has remarkable flexibility in its positioning as a transcription activator and that different interactions between ToxT and RNA polymerase occur during transcription activation of promoters having different toxbox configurations.The gram-negative bacterium Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, continues to be a serious public health problem despite over 100 years of research on its pathogenesis. The current cholera pandemic, which began in 1961, continues unabated, and an estimated 5 million cases of cholera occur worldwide each year. Although over 200 known serogroups of V. cholerae have been identified, only the O1 and O139 serogroups are capable of causing pandemic cholera (34,35).Pandemic V. cholerae strains require two major virulence factors for colonization and pathogenesis. The first factor, cholera toxin, is an A 1 B 5 -type ADP-ribosylating toxin that is responsible for producing the voluminous watery diarrhea characteristic of cholera (8,26). The genes encoding cholera toxin, ctxAB, are carried in the genome of a lysogenic bacteriophage, CTX⌽ (40). The second major virulence factor is the toxin-coregulated pilus (TCP) (38), a type IV pilus that initiates microcolony formation and is required for intestinal colonization by V. cholerae (39). Genes encoding the TCP, which are in a long operon beginning with the tcpA pilin gene, are located in the Vibrio pathogenicity island (VPI) (22). In addition to its role as a colonization factor, the TCP is also the receptor for CTX⌽ and thus allows horizontal transfer of ctxAB to nontoxigenic V. cholerae carrying the VPI (40). In addition to the TCP-encoding genes, several other genes thought to...

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Section: Discussionmentioning

confidence: 87%

Flexibility of Vibrio cholerae ToxT in Transcription Activation of Genes Having Altered Promoter Spacing

Bellair

Withey

2008

J Bacteriol

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“…An NMR study on mixtures of a MarA-marbox complex with the ␣CTD of RNAP indicates that Trp19 and Arg36 of MarA interact with ␣CTD at a "265-like DNA-binding determinant" of ␣CTD; the Trp19Ala and Arg36Ala mutations only minimally affect binding to the mar marbox in EMSA assays (4). Furthermore, the Trp19Ala mutation of MarA decreases binding in vitro of the MarA-marbox to the ␣CTD and is proposed therefore to reduce prerecruitment of MarA by RNAP (4).…”

Section: Resultsmentioning

confidence: 99%

Evidence that Regulatory Protein MarA of Escherichia coli Represses rob by Steric Hindrance

McMurry¹,

Levy

2010

J Bacteriol

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The MarA protein of Escherichia coli can both activate and repress the initiation of transcription, depending on the position and orientation of its degenerate 20-bp binding site ("marbox") at the promoter. For all three known repressed genes, the marbox overlaps the promoter. It has been reported that MarA represses the rob promoter via an RNA polymerase (RNAP)-DNA-MarA ternary complex. Under similar conditions, we found a ternary complex for the repressed purA promoter also. These findings, together with the backwards orientation of repressed marboxes, suggested a unique interaction of MarA with RNAP in repression. However, no repression-specific residues of MarA could be found among 38 single-alanine replacement mutations previously shown to retain activation function or among mutants from random mutagenesis. Mutations Thr12Ala, Arg36Ala, Thr95Ile, and Pro106Ala were more damaging for activation than for repression, some up to 10-fold, so these residues may play a specific role in activation. We found that nonspecific binding of RNAP to promoterless regions of DNA was presumably responsible for the ternary complexes seen previously. When RNAP binding was promoter specific, MarA reduced RNAP access to the rob promoter; there was little or no ternary complex. These findings strongly implicate steric hindrance as the mechanism of repression of rob by MarA.

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“…This capacity is related to the function of the ␣CTD, because all the mutations found in this study were mapped to this region. This domain of the protein has been implicated in contacting promoter DNA and protein activators and repressors (6,9), suggesting that it is the regulatory function of the alpha subunit which gives rise to the pleiotropic alteration of the transcriptome that results in novel phenotypes. The ␣NTD has also been shown to be a target for transcription regulation (32), but no mutations were found there in the present study.…”

Section: Discussionmentioning

confidence: 99%

Mutagenesis of the Bacterial RNA Polymerase Alpha Subunit for Improvement of Complex Phenotypes

Klein‐Marcuschamer

Santos

et al. 2009

Appl Environ Microbiol

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Combinatorial or random methods for strain engineering have been extensively used for the improvement of multigenic phenotypes and other traits for which the underlying mechanism is not fully understood. Although the preferred method has traditionally been mutagenesis and selection, our laboratory has successfully used mutant transcription factors, which direct the RNA polymerase (RNAP) during transcription, to engineer complex phenotypes in microbial cells. Here, we show that it is also possible to impart new phenotypes by altering the RNAP core enzyme itself, in particular through mutagenesis of the alpha subunit of the bacterial polymerase. We present the use of this tool for improving tolerance of Escherichia coli to butanol and other solvents and for increasing the titers of two commercially relevant products, L-tyrosine and hyaluronic acid. In addition, we explore the underlying physiological changes that give rise to the solvent-tolerant mutant.

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Versatility of the carboxy‐terminal domain of the α subunit of RNA polymerase in transcriptional activation: use of the DNA contact site as a protein contact site for MarA

Cited by 47 publications

References 57 publications

Flexibility of Vibrio cholerae ToxT in Transcription Activation of Genes Having Altered Promoter Spacing

Flexibility of Vibrio cholerae ToxT in Transcription Activation of Genes Having Altered Promoter Spacing

Evidence that Regulatory Protein MarA of Escherichia coli Represses rob by Steric Hindrance

Mutagenesis of the Bacterial RNA Polymerase Alpha Subunit for Improvement of Complex Phenotypes

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