Repression of Transcription Initiation at 434 PR by 434 Repressor: Effects on Transition of a Closed to an Open Promoter Complex

Xu, Jian; Koudelka, Gerald B.

doi:10.1006/jmbi.2001.4702

Cited by 12 publications

(18 citation statements)

References 33 publications

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“…In bacteriophage , Ͼ20-fold more repressor is required to repress P RM transcription from an O R template than is needed to maximally stimulate this promoter's activity (17,29). Similar results were obtained with bacteriophage 434 (10,43).…”

Section: Wsupporting

confidence: 86%

The Lysis-Lysogeny Decision of Bacteriophage 933W: a 933W Repressor-Mediated Long-Distance Loop Has No Role in Regulating 933W P _RM Activity

Bullwinkle

Koudelka

2011

J Bacteriol

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Our data show that unlike bacteriophage , repressor bound at O L of bacteriophage 933W has no role in regulation of 933W repressor occupancy of 933W O R 3 or the transcriptional activity of 933W P RM . This finding suggests that a cooperative long-range loop between repressor tetramers bound at O R and O L does not form in bacteriophage 933W. Nonetheless, 933W forms lysogens, and 933W prophage display a threshold response to UV induction similar to related lambdoid phages. Hence, the long-range loop thought to be important for constructing a threshold response in lambdoid bacteriophages is dispensable. The lack of a loop requires bacteriophage 933W to use a novel strategy in regulating its lysis-lysogeny decisions. As part of this strategy, the difference between the repressor concentrations needed to bind O R 2 and activate 933W P RM transcription or bind O R 3 and repress transcription from P RM is <2-fold. Consequently, P RM is never fully activated, reaching only ϳ25% of the maximum possible level of repressor-dependent activation before repressormediated repression occurs. The 933W repressor also apparently does not bind cooperatively to the individual sites in O R and O L . This scenario explains how, in the absence of DNA looping, bacteriophage 933W displays a threshold effect in response to DNA damage and suggests how 933W lysogens behave as "hair triggers" with spontaneous induction occurring to a greater extent in this phage than in other lambdoid phages.Studies of how the lysis-lysogeny decisions of lambdoid bacteriophages are regulated have illustrated the importance of short-and long-range cooperative DNA binding by proteins in controlling a complex gene regulatory network. In all lambdoid phages, the repressor protein directs the establishment and maintenance of the lysogenic state by simultaneously repressing transcription of the genes needed for lytic phage growth and activating transcription of a gene needed for lysogen formation (30). Based primarily on studies of bacteriophage , two different cooperative repressor-DNA binding events are thought to be required for regulation of lambdoid phage lysogen development. The first involves formation of a repressor tetramer between two repressor dimers, one bound to each of the adjacent O R 1 and O R 2 sites. A similar tetramer is also formed at the O L 1 and O L 2 sites. The "side-by-side" cooperative binding by a repressor tetramer is prerequisite to forming a stable lysogens (2,20,30).Additional cooperative interactions between two tetramers, each bound to a pair of adjacent operators that are separated by Ͼ2.5 kb, occur in bacteriophages and P22 (13-15, 31). In , a repressor octamer-mediated O L -cI 8 -O R complex forms with the intervening DNA looping out (3,13,15). This longrange cooperative interaction helps modulate the prophage's compensatory response to low doses of DNA damage (3) and thereby regulates lysogen stability. In all well-studied lambdoid phages, the amount of phage produced is not linearly related to the amount of DNA damage until ...

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Section: Wsupporting

confidence: 86%

The Lysis-Lysogeny Decision of Bacteriophage 933W: a 933W Repressor-Mediated Long-Distance Loop Has No Role in Regulating 933W P _RM Activity

Bullwinkle

Koudelka

2011

J Bacteriol

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“…Nonetheless, 434 repressor binding to O R 2 is necessary and sufficient to prevent transcription from P R . Thus, in phage 434, the O R 2-bound repressor blocks transcription initiation by blocking the transition of a P R -bound RNA polymerase from a stable closed complex to an open complex (52). The position- ing of the P R promoter elements with respect to the 933W repressor binding sites in O R resembles that found in bacteriophage 434, suggesting that 933W repressor represses initiation of transcription at P R using a mechanism similar to that used by 434 repressor.…”

Section: Discussionmentioning

confidence: 87%

Purification and Characterization of the Repressor of the Shiga Toxin-Encoding Bacteriophage 933W: DNA Binding, Gene Regulation, and Autocleavage

2004

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The genes encoding Shiga toxin (stx), the major virulence factor of Shiga toxin-encoding Escherichia coli (STEC) strains, are carried on lambdoid prophages resident in all known STEC strains. The stx genes are expressed only during lytic growth of these temperate bacteriophages. We cloned the gene encoding the repressor of the Shiga toxin-encoding bacteriophage 933W and examined the DNA binding and transcriptional regulatory activities of the overexpressed, purified protein. Typical of nearly all lambdoid phage repressors, 933W repressor binds to three sites in 933W right operator (O R ). Also typical, when bound at O R , 933W repressor functions as an activator at the P RM promoter and a repressor at the P R promoter. In contrast to other lambdoid bacteriophages, 933W left operator (O L ) contains only two repressor binding sites, but the O L -bound repressor still efficiently represses P L transcription. Lambdoid prophage induction requires inactivation of the repressor's DNA binding activity. In all phages examined thus far, this inactivation requires a RecA-stimulated repressor autoproteolysis event, with cleavage occurring precisely in an Ala-Gly dipeptide sequence that is found within a "linker " region that joins the two domains of these proteins. However, 933W repressor protein contains neither an Ala-Gly nor an alternative Cys-Gly dipeptide cleavage site anywhere in its linker sequence. We show here that the autocleavage occurs at a Leu-Gly dipeptide. Thus, the specificity of the repressor autocleavage site is more variable than thought previously.Shiga toxins (stx) are the major virulence factors in enterohemorrhagic Escherichia coli infections, causing such diseases as hemorrhagic colitis, infantile diarrhea, and hemolytic uremic syndrome. In virtually all known Shiga toxin-encoding E. coli strains, the genes encoding Shiga toxins are carried on lambdoid prophages (8,19,31,34,35,45) as part of an operon whose activity is ultimately regulated by the bacteriophage repressor (32,33,46,47).Lambdoid phage genomes contain two operator regions, O L and O R , each of which includes promoters whose expression is controlled by the binding of the bacteriophage repressor to multiple binding sites found in each operator region. Efficient functioning of the genetic switch between lysis and lysogeny depends on the ability of the repressor to bind with different affinities to each of the individual sites within O R and O L (40). The repressor directs the establishment and maintenance of the lysogenic state by simultaneously repressing transcription of the genes needed for lytic phage growth and activating transcription of its own gene, the only gene needed for maintenance of the lysogenic state (40).The E. coli O157:H7 strain EDL933 is considered to be the reference strain for disease-causing O157:H7 isolates. Incubating this strain with agents that trigger induction of resident prophages causes this strain to express the disease-causing stx2 gene product and to produce a lambdoid bacteriophage (38). Sequence analysi...

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“…This region has been called the "exclusive zone of repression" (30), as transcription factorbinding sites that lie within this position almost always lead to repression, e.g. IclR at the iclR promoter (31), Fur at the aerobactin promoter (32), and the 434 repressor at 434 bacteriophage P r promoter (33). Exceptions to this rule do exist such as positive regulation by MerR at the merTPAD promoter (34), Arc at the P ant variant promoter of bacteriophage P22 (35), and SoxR at the soxS promoter (36).…”

Section: Discussionmentioning

confidence: 99%

“…Because helical dependence is a critical factor in activation by MarA (19), the transcriptional outcome, repression versus activation, may be dependent on which DNA face the protein binds as is the case for GalR (41). The phage 434 protein represses the P r promoter via a binding site that lies between the Ϫ10 and Ϫ35 hexamers (33,42). Ethylation interference experiments at this promoter demonstrate that repression occurs via the binding of the 434 repressor and RNAP on opposite faces of the promoter (33).…”

Section: Discussionmentioning

confidence: 99%

“…The phage 434 protein represses the P r promoter via a binding site that lies between the Ϫ10 and Ϫ35 hexamers (33,42). Ethylation interference experiments at this promoter demonstrate that repression occurs via the binding of the 434 repressor and RNAP on opposite faces of the promoter (33). Alternatively, specific interactions between MarA and the rob promoter may be different from that described for the activated promoters (28) and occur through only one of the helix-turn-helix motifs as predicted in the Rob- micF crystal structure, thereby allowing both proteins to bind the same face of the DNA (8).…”

Section: Discussionmentioning

confidence: 99%

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MarA-mediated Transcriptional Repression of the rob Promoter

Schneiders

Levy

2006

Journal of Biological Chemistry

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The Escherichia coli transcriptional regulator MarA affects functions that include antibiotic resistance, persistence, and survival. MarA functions as an activator or repressor of transcription utilizing similar degenerate DNA sequences (marboxes) with three different binding site configurations with respect to the RNA polymerase-binding sites. We demonstrate that MarA down-regulates rob transcripts both in vivo and in vitro via a MarA-binding site within the rob promoter that is positioned between the ؊10 and ؊35 hexamers. As for the hdeA and purA promoters, which are repressed by MarA, the rob marbox is also in the "backward" orientation. Protein-DNA interactions show that SoxS and Rob, like MarA, bind the same marbox in the rob promoter. Electrophoretic mobility shift analyses with a MarA-specific antibody demonstrate that MarA and RNA polymerase form a ternary complex with the rob promoter DNA. Transcription experiments in vitro and potassium permanganate footprinting analysis show that MarA affects the RNA polymerase-mediated closed to open complex formation at the rob promoter.The Rob protein is an abundant nucleoid protein that was initially discovered bound to the right origin of replication of the Escherichia coli chromosome (1). Despite its association with the origin of replication, no experimental evidence supports the role of Rob in chromosome replication, chromatin structure, and superhelicity (2).Rob is, however, a member of a subgroup within the AraC/XylS family of transcriptional regulators, which includes the MarA and SoxS proteins that are involved in a wide range of regulatory functions (3). The N-terminal domain of Rob shares similarity with MarA and SoxS, which is in contrast to the other members of the subgroup that share homology within the C-terminal domain (3). When induced by bile salts and dipyridyl, the C-terminal domain undergoes post-transcriptional modification that results in the conversion of Rob from a low to a high activity state in the cell (4). However, in the absence of these compounds, overexpression of either the full or C-terminal domain deleted protein is sufficient for activity and promoter binding in vitro (5, 6) and in vivo (5, 7). This observation is consistent with structural data derived from the Rob-micF complex, where only the N-terminal domain makes DNA-specific contacts (8). Overexpression of rob confers multidrug, organic solvent and heavy metal resistance (6, 7); in accord, some experimental data indicate that rob null mutants are hypersensitive to antibiotics, organic solvents, and heavy metals (5, 7, 9).Transposon mutagenesis experiments aimed at defining the Rob regulon revealed eight namely inaA, marRAB, aslB, ybaO, mdlA, yfhD, ybiS, and galT (10), some of which are also known members of the mar and sox regulons (11). In addition, studies demonstrate that Rob induced by decanoate or bile salts results in the increased expression of acrAB in the absence of both the mar and sox loci (12).The expression levels of rob do not vary dramatically during differ...

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Repression of Transcription Initiation at 434 PR by 434 Repressor: Effects on Transition of a Closed to an Open Promoter Complex

Cited by 12 publications

References 33 publications

The Lysis-Lysogeny Decision of Bacteriophage 933W: a 933W Repressor-Mediated Long-Distance Loop Has No Role in Regulating 933W P _RM Activity

The Lysis-Lysogeny Decision of Bacteriophage 933W: a 933W Repressor-Mediated Long-Distance Loop Has No Role in Regulating 933W P _RM Activity

Purification and Characterization of the Repressor of the Shiga Toxin-Encoding Bacteriophage 933W: DNA Binding, Gene Regulation, and Autocleavage

MarA-mediated Transcriptional Repression of the rob Promoter

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Repression of Transcription Initiation at 434 PR by 434 Repressor: Effects on Transition of a Closed to an Open Promoter Complex

Cited by 12 publications

References 33 publications

The Lysis-Lysogeny Decision of Bacteriophage 933W: a 933W Repressor-Mediated Long-Distance Loop Has No Role in Regulating 933W P RM Activity

The Lysis-Lysogeny Decision of Bacteriophage 933W: a 933W Repressor-Mediated Long-Distance Loop Has No Role in Regulating 933W P RM Activity

Purification and Characterization of the Repressor of the Shiga Toxin-Encoding Bacteriophage 933W: DNA Binding, Gene Regulation, and Autocleavage

MarA-mediated Transcriptional Repression of the rob Promoter

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The Lysis-Lysogeny Decision of Bacteriophage 933W: a 933W Repressor-Mediated Long-Distance Loop Has No Role in Regulating 933W P _RM Activity

The Lysis-Lysogeny Decision of Bacteriophage 933W: a 933W Repressor-Mediated Long-Distance Loop Has No Role in Regulating 933W P _RM Activity