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

Dhiman, Anjali; Schleif, Robert

doi:10.1128/jb.182.18.5076-5081.2000

Cited by 21 publications

(16 citation statements)

References 47 publications

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“…The bars indicate standard errors. ATG transcript initiation point would place bound C.PvuII at a rare but by no means unique position for transcriptional activators (17,32,48,52,63). In addition, a fortuitous region of very high similarity between C.PvuII and the cI repressoractivator of bacteriophage (Fig.…”

Section: Discussionmentioning

confidence: 99%

Nature of the Promoter Activated by C.PvuII, an Unusual Regulatory Protein Conserved among Restriction-Modification Systems

et al. 2005

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A widely distributed family of small regulators, called C proteins, controls a subset of restriction-modification systems. The C proteins studied to date activate transcription of their own genes and that of downstream endonuclease genes; this arrangement appears to delay endonuclease expression relative to that of the protective methyltransferase when the genes enter a new cell. C proteins bind to conserved sequences called C boxes. In the PvuII system, the C boxes have been reported to extend from ؊23 to ؉3 relative to the transcription start for the gene for the C protein, an unexpected starting position relative to a bound activator. This study suggests that transcript initiation within the C boxes represents initial, C-independent transcription of pvuIICR. The major C protein-dependent transcript appears to be a leaderless mRNA starting farther downstream, at the initiation codon for the pvuIIC gene. This conclusion is based on nuclease S1 transcript mapping and the effects of a series of nested deletions in the promoter region. Furthermore, replacing the region upstream of the pvuIIC initiation codon with a library of random oligonucleotides, followed by selection for C-dependent transcription, yielded clones having sequences that resemble ؊10 promoter hexamers. The ؊35 hexamer of this promoter would lie within the C boxes. However, the spacing between C boxes/؊35 and the apparent ؊10 hexamer can be varied by ؎4 bp with little effect. This suggests that, like some other activator-dependent promoters, PpvuIICR may not require a ؊35 hexamer. Features of this transcription activation system suggest explanations for its broad host range.The genera hosting restriction-modification (RM) systems appear to comprise the majority of the prokaryotic world. RM systems play a variety of roles, ranging from the simply selfish (44, 54) through defense against bacteriophages (42, 51) to facilitating recombination (34, 36). While much remains to be learned about the functions of RM systems, our understanding of how they are regulated is even more limited. Most, if not all, RM system genes can move from cell to cell, either by residing on a plasmid or because of transduction, transformation, or Hfr-type conjugation. As a result, they all face a critical regulatory problem-how to ensure that a new host's DNA is protectively methylated before endonuclease activity appears. This problem is particularly acute for the type II RM systems, in which the methyltransferase (MTase) and endonuclease (REase) function independently.A subset of type II RM systems includes, in addition to genes for the MTase and REase, a third gene for a conserved regulator called the C (controller) protein. C proteins were originally discovered in the PvuII (58, 59) and BamHI (22, 57) RM systems and were then noted in several other systems as well

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Section: Discussionmentioning

confidence: 99%

Nature of the Promoter Activated by C.PvuII, an Unusual Regulatory Protein Conserved among Restriction-Modification Systems

et al. 2005

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“…Evidence for AraC interactions with 70 come from early 70 mutations (eventually identified at R596) that increased araBAD expression in the absence of activation by cyclic AMP receptor protein (CRP) (18,39,44), as well as the finding that araBAD expression in a ⌬cya strain was increased by 70 E591A and R596A and decreased by 70 K593A (27). In addition, with a DNA that mimicked an open complex, a small amount of DNA-binding cooperativity could be detected between AraC and 70 (7). At the melAB promoter, genetic evidence indicated that 70 R596 interacts with MelR D261 and T265 while 70 R599 also interacts with MelR D261, which aligns with RhaS D241 (16).…”

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confidence: 90%

Amino Acid Contacts between Sigma 70 Domain 4 and the Transcription Activators RhaS and RhaR

Wickstrum

Egan

2004

J Bacteriol

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The RhaS and RhaR proteins are transcription activators that respond to the availability of L-rhamnose and activate transcription of the operons in the Escherichia coli L-rhamnose catabolic regulon. RhaR activates transcription of rhaSR, and RhaS activates transcription of the operon that encodes the L-rhamnose catabolic enzymes, rhaBAD, as well as the operon that encodes the L-rhamnose transport protein, rhaT. RhaS is 30% identical to RhaR at the amino acid level, and both are members of the AraC/XylS family of transcription activators. The RhaS and RhaR binding sites overlap the ؊35 hexamers of the promoters they regulate, suggesting they may contact the 70 subunit of RNA polymerase as part of their mechanisms of transcription activation. In support of this hypothesis, our lab previously identified an interaction between RhaS residue D241 and 70 residue R599. In the present study, we first identified two positively charged amino acids in 70 , K593 and R599, and three negatively charged amino acids in RhaR, D276, E284, and D285, that were important for RhaR-mediated transcription activation of the rhaSR operon. Using a genetic loss-of-contact approach we have obtained evidence for a specific contact between RhaR D276 and 70 R599. Finally, previous results from our lab separately showed that RhaS D250A and 70 K593A were defective at the rhaBAD promoter. Our genetic loss-of-contact analysis of these residues indicates that they identify a second site of contact between RhaS and 70 .Transcription activation in Escherichia coli often involves the interaction of a DNA-binding activator protein with one of the subunits of RNA polymerase (RNAP), most often the sigma () or alpha (␣) subunit. Transcription activators that bind immediately upstream and adjacent to RNAP, in some cases overlapping the Ϫ35 promoter hexamer, may interact with the C-terminal domain (domain 4) of the subunit of RNAP (8,27). The cI protein of bacteriophage is required for the establishment and maintenance of lysogeny and is perhaps the best-characterized example of a transcription activator that contacts 70 . The cI protein activates transcription of the P RM promoter when bound at the O R 2 operator site, which overlaps the P RM Ϫ35 hexamer by 2 bp (30). Current evidence suggests that 70 residues R588, K593, and R596 are required for activation by cI (23,26,35). Genetic and molecular modeling studies, as well as the recent structure of a ternary cI-domain 4-DNA complex, indicate that cI D38 contacts both 70 K593 ( A K418) and R596 ( A R421) and cI E34 contacts 70 R588 ( A R413) (8,19,26,35). Prior to the identification of the ternary complex structure, a molecular model of the interaction indicated that 70 K593 ( A K418) contacts DNA but was not positioned to contact cI (6,8,35). However, the ternary structure showed that the A residue that aligns with 70 K593 has moved away from the DNA (relative to the model of the interaction) and instead makes a proteinprotein contact with cI D38 (19).There is also evidence that activation by several tran...

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“…A classic example is that of the pho operon, where the PhoB binding site is centered at 2 40, occupying what would have been the 2 35 box. 13,14 Other studies, such as string search analysis, 4 revealed a third element upstream from the 2 35 box, the UP element, originally associated with "bent DNA", 15 and more recently associated with the binding region of the carboxy-terminal domain of the alpha subunit of RNAP. 16 In about 3% of the E. coli promoters, the UP element is located approximately 4 bp upstream of the 2 35 region conferring additional strength to the promoter.…”

Section: Introductionmentioning

confidence: 99%

Sigma70 Promoters in Escherichia coli: Specific Transcription in Dense Regions of Overlapping Promoter-like Signals

Huerta¹,

Collado‐Vides²

2003

Journal of Molecular Biology

218

227

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

Cited by 21 publications

References 47 publications

Nature of the Promoter Activated by C.PvuII, an Unusual Regulatory Protein Conserved among Restriction-Modification Systems

Nature of the Promoter Activated by C.PvuII, an Unusual Regulatory Protein Conserved among Restriction-Modification Systems

Amino Acid Contacts between Sigma 70 Domain 4 and the Transcription Activators RhaS and RhaR

Sigma70 Promoters in Escherichia coli: Specific Transcription in Dense Regions of Overlapping Promoter-like Signals

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