AraC Protein Can Activate Transcription from Only One Position and When Pointed in Only One Direction

Reeder, Thadd; Schleif, Robert

doi:10.1006/jmbi.1993.1276

Cited by 55 publications

(75 citation statements)

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“…Despite these differences, one identically oriented ExsA consensus element is found at bp Ϫ51 or Ϫ52 with respect to the transcriptional start sites of pD, pS, and pORF1. This common location is similar to the specific orientation and distance requirements for the AraC consensus binding sites (38).…”

Section: Discussionsupporting

confidence: 64%

Analyses of the DNA-binding and transcriptional activation properties of ExsA, the transcriptional activator of the Pseudomonas aeruginosa exoenzyme S regulon

1995

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ExsA has been implicated as a central regulator of exoenzyme S production by Pseudomonas aeruginosa. In this study, the DNA-binding and transcriptional activation properties of ExsA were investigated. ExsA was produced and purified as a fusion protein, MALA3A2, which was shown to bind specifically to promoter regions that regulated transcription of the exoenzyme S trans-regulatory locus (pC) and a locus located directly downstream of exsA (pD). Previously, MALA3A2 was shown to bind the exoS 5 PstI-NsiI region, which contained two independent but coordinately regulated (ExsA-mediated) promoters, pS (now termed pORF1) and pS. DNase I footprint analysis of the promoter regions bound by ExsA revealed a common protected consensus sequence of TXAAAAXA. The consensus sequence was located ؊51 to ؊52 bp upstream of the transcriptional start sites for pD, pS, and pORF1. Promoter fusion, DNA-binding, and mutagenesis analyses indicated that the consensus sequence was important for transcriptional activation. Each ExsA-controlled promoter region contained at least two consensus sites in close proximity, similar to the arrangement of half-sites seen in AraC-controlled (Escherichia coli) or VirF-controlled (Yersinia enterocolitica) promoters. However, the results of this study suggested that only one consensus site was required in the exoenzyme S (pS) or ORF1 promoter (pORF1) to initiate transcription. These data suggest that members of the exoenzyme S regulon can be defined as possessing an ExsA consensus element which maps at bp ؊51 or ؊52 relative to the transcriptional start site.

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Section: Discussionsupporting

confidence: 64%

Analyses of the DNA-binding and transcriptional activation properties of ExsA, the transcriptional activator of the Pseudomonas aeruginosa exoenzyme S regulon

1995

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“…The binding of AraC to the I 1 and I 2 half-sites is stimulated by the presence of arabinose. When these sites are occupied by AraC, and if they overlap the Ϫ35 hexamer by 2 or 4 bases, transcription is actively initiated from p BAD (39).…”

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confidence: 99%

Recognition of Overlapping Nucleotides by AraC and the Sigma Subunit of RNA Polymerase

Dhiman

Schleif

2000

J Bacteriol

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The Escherichia coli promoter p BAD , under the control of the AraC protein, drives the expression of mRNA encoding the AraB, AraA, and AraD gene products of the arabinose operon. The binding site of AraC at p BAD overlaps the RNA polymerase ؊35 recognition region by 4 bases, leaving 2 bases of the region not contacted by AraC. This overlap raises the question of whether AraC substitutes for the sigma subunit of RNA polymerase in recognition of the ؊35 region or whether both AraC and sigma make important contacts with the DNA in the ؊35 region. If sigma does not contact DNA near the ؊35 region, p BAD activity should be independent of the identity of the bases in the hexamer region that are not contacted by AraC. We have examined this issue in the p BAD promoter and in a second promoter where the AraC binding site overlaps the ؊35 region by only 2 bases. In both cases promoter activity is sensitive to changes in bases not contacted by AraC, showing that despite the overlap, sigma does read DNA in the ؊35 region. Since sigma and AraC are thus closely positioned at p BAD , it is possible that AraC and sigma contact one another during transcription initiation. DNA migration retardation assays, however, showed that there exists only a slight degree of DNA binding cooperativity between AraC and sigma, thus suggesting either that the normal interactions between AraC and sigma are weak or that the presence of the entire RNA polymerase is necessary for significant interaction.The sigma subunit of RNA polymerase (referred to here as sigma) is responsible for the binding of the holoenzyme to promoters during transcription initiation (2, 46). It does this by making sequence-specific contacts with bases in hexameric sequences centered at 10 and 35 bases upstream of the transcription start site on promoters (3,13,18,32,45,50). At the Ϫ10 hexamer, sigma makes base-specific contacts with the nontemplate strand (23,34,41,42). In addition to sigma-DNA interactions during initiation, protein-protein contacts also occur between transcriptional activators and subunits of RNA polymerase (1,11,14,21,22,36,43).At many promoters, the recognition sequences of transcriptional activator proteins partly overlap the 6 bases of the Ϫ35 region that are contacted by the sigma subunit of RNA polymerase (4). In these cases, does the activator substitute for sigma in the recognition of the Ϫ35 region; do both proteins read the Ϫ35 region, necessitating overlapped reading by both proteins; or does sigma read an adjacent sequence?On one hand, direct protein-protein contacts between sigma and upstream transcriptional activators seem to occur. At the p RM promoter, the binding site of cI overlaps the Ϫ35 region for sigma by 2 nucleotides, and genetic experiments suggest an interaction between the cI protein and the Ϫ35 recognition motif of sigma 70 (25, 31). Recently, interactions between sigma and Ada, an AraC homologue from the XylS family of proteins, have been demonstrated genetically at the ada, alkA, and aidB promoters (27, 28). A direct sigma-A...

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“…The loop structure also suppresses araC transcription, which occurs in the opposite direction from that of araBAD (11,14). When arabinose is present, the binding of a monomeric AraC shifts to araI 2 (located toward P BAD as part of araI) from araO 2 , thereby activating the transcription of P BAD (3,20,29,33). The cyclic AMP receptor protein (CRP) plays a role by binding to a region upstream of araI, thereby opening the loop to initiate transcription from both P BAD and P C (21).…”

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confidence: 99%

Organization and regulation of the D-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator

1997

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The metabolism of D-xylose in Escherichia coli K-12 is known to be mediated by the xylAB gene. However, the nearby xylFGHR genes were found by genome sequencing and predicted to be responsible for transport and regulation for xylose based on their sequence similarities to other functionally related genes. Here, we investigated transcriptional organization and functions of the xyl genes. An analysis with random transposon insertions revealed that the xyl genes are organized into two major transcriptional units, xylAB and xylFGHR, governed by the promoters P A and P F , respectively. However, there is an additional weak promoter, P R , which is specific for xylR. Sites of transcription initiation were determined by primer extension analysis. When studied with operon fusions to lacZ, the P A and P F promoters were activated by D-xylose and repressed by glucose. In contrast, the P R promoter was not regulated by these sugars. A mutation in xylR completely abolished expression from the P A and P F promoters, causing a defect in both growth and transport.

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AraC Protein Can Activate Transcription from Only One Position and When Pointed in Only One Direction

Cited by 55 publications

References 0 publications

Analyses of the DNA-binding and transcriptional activation properties of ExsA, the transcriptional activator of the Pseudomonas aeruginosa exoenzyme S regulon

Analyses of the DNA-binding and transcriptional activation properties of ExsA, the transcriptional activator of the Pseudomonas aeruginosa exoenzyme S regulon

Recognition of Overlapping Nucleotides by AraC and the Sigma Subunit of RNA Polymerase

Organization and regulation of the D-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator

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