Escherichia coli single-stranded DNA-binding protein is a supercoiled template-dependent transcriptional activator of N4 virion RNA polymerase.

Markiewicz, Peter; Malone, C.; Chase, John W.; Rothman-Denes, Lucia B.

doi:10.1101/gad.6.10.2010

Cited by 38 publications

(25 citation statements)

References 69 publications

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“…vRNAP-promoter recognition is the first event after injection of the genome into the host. We have suggested that the dependence of N4 early RNA synthesis on gyrase activity allows the phage to monitor the ability of the host cell to support phage growth (15), since the level of DNA supercoiling inside the cell is influenced by the [ATP]͞[ADP] ratio (42)(43)(44). On the other hand, hairpin extrusion could inhibit the binding of sequence-specific DNA binding proteins to their cognate sites if these sites overlap extruding sequences.…”

Section: Discussionmentioning

confidence: 99%

“…Mutational analyses of the promoter template strands indicated that specific sequences and a 5-to 7-base-pair stem, 3-base loop hairpin are important for transcriptional activity (14). Transcription on doublestranded DNA requires supercoiling and Escherichia coli single-stranded DNA-binding protein (EcoSSB) (15). On the basis of these results, we proposed a model in which negative supercoiling extrudes a hairpin to yield an active promoter conformation that is recognized by N4 vRNAP (14).…”

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confidence: 99%

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Supercoil-induced extrusion of a regulatory DNA hairpin

Dai

Greizerstein

Nadas-Chinni

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Bacteriophage N4 virion RNA polymerase (N4 vRNAP) promoters contain inverted repeats, which form a 5-to 7-base-pair stem, 3-base loop hairpin that is required for vRNAP recognition. We show that, contrary to certain theoretical predictions, hairpin extrusion can occur at physiological superhelical densities in a Mg 2؉ -dependent manner. Specific sequences on the template strand are required for hairpin extrusion. These sequences define stable DNA hairpins that are relatively unreactive to single strand-specific probes. In addition, a specific stable hairpin-inducing sequence can regulate transcription in vivo. Thus, a DNA structure, in its natural environment, is involved in transcriptional regulation.DNA supercoiling affects transcription by facilitating or inhibiting RNA polymerase (RNAP) binding and͞or formation of the open complex (1-3). In addition, by stabilizing DNA bending and͞or looping (4-7), supercoiling enhances or inhibits the interaction of activators or repressors with the transcriptional machinery. Supercoiling also promotes the formation of noncanonical DNA structures such as cruciforms, Z-DNA, or triple helices (8) which can affect transcription in vitro (9-11). In no instance, however, is there convincing evidence that such structures play a role in vivo in regulating transcription (reviewed in ref. 12).The three bacteriophage N4 early promoters utilized by the virion (v)RNAP share sequence from Ϫ18 to ϩ1 containing small inverted repeats centered at Ϫ12 (ref. 13). On singlestranded DNA templates, these promoters are utilized efficiently by N4 vRNAP (13,14). Mutational analyses of the promoter template strands indicated that specific sequences and a 5-to 7-base-pair stem, 3-base loop hairpin are important for transcriptional activity (14). Transcription on doublestranded DNA requires supercoiling and Escherichia coli single-stranded DNA-binding protein (EcoSSB) (15). On the basis of these results, we proposed a model in which negative supercoiling extrudes a hairpin to yield an active promoter conformation that is recognized by N4 vRNAP (14). On the contrary, the current theory of the energetics of hairpin extrusion predicts that such small hairpins will extrude only at high, unphysiological superhelical densities (16,17). Here, we show that extrusion of N4 early promoter hairpins occurs at physiological superhelical densities, that this event requires Mg 2ϩ and specific sequence, and that extrusion occurs in vivo. MATERIALS AND METHODS Preparation of Circles Containing N4 Early Promoters.Cloning of the 2.2-kb SpeI-PstI fragment, containing the N4 early promoters P1 and P2 followed by their respective terminators t1 and t2, into pKB652 to yield pXD102, was as described (18). To generate mutant P1 promoters, pXD102 was digested with SmaI and partially digested with NsiI; the 4844-bp fragment lacking the P1 promoter was ligated to the M13 mp11 SmaI-PstI fragment carrying the wild-type or mutant P1 promoters (14). In vivo generation of circles, isolation, preparation of topoisomers, and d...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Supercoil-induced extrusion of a regulatory DNA hairpin

Dai

Greizerstein

Nadas-Chinni

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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“…In vivo, N4 early transcription requires Escherichia coli DNA gyrase and E. coli ssDNA-binding protein (EcoSSB), which provide an ''active'' promoter structure (5). Other SSBs (T7 gp 2.5, T4 gp 32, and N4 SSB) fail to stimulate vRNAP transcription in vitro because they destabilize the promoter hairpin (6).…”

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confidence: 99%

Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase

Davydova

Rothman-Denes

2003

Proc. Natl. Acad. Sci. U.S.A.

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Coliphage N4 virion RNA polymerase (vRNAP), the most distantly related member of the T7-like family of RNA polymerases, is responsible for transcription of the early genes of the linear double-stranded DNA phage genome. Escherichia coli singlestranded DNA-binding protein (EcoSSB) is required for N4 early transcription in vivo, as well as for in vitro transcription on supercoiled DNA templates containing vRNAP promoters. In contrast to other DNA-dependent RNA polymerases, vRNAP initiates transcription on single-stranded, promoter-containing templates with in vivo specificity; however, the RNA product is not displaced, thus limiting template usage to one round. We show that EcoSSB activates vRNAP transcription at limiting single-stranded template concentrations through template recycling. EcoSSB binds to the template and to the nascent transcript and prevents the formation of a transcriptionally inert RNA:DNA hybrid. Using C-terminally truncated EcoSSB mutant proteins, human mitochondrial SSB (Hsmt SSB), phage P1 SSB, and F episome-encoded SSB, as well as a Hsmt-EcoSSB chimera, we have mapped a determinant of template recycling to the C-terminal amino acids of EcoSSB. T7 RNAP contains an amino-terminal domain responsible for binding the RNA product as it exits from the enzyme. No sequence similarity to this domain exists in vRNAP. Hereby, we propose a unique role for EcoSSB: It functionally substitutes in N4 vRNAP for the N-terminal domain of T7 RNAP responsible for RNA binding.B acteriophage N4 virion RNA polymerase (vRNAP), responsible for transcription of the phage early genes, is injected into the host cell with the phage genome upon infection (1). vRNAP is inactive on linear double-stranded templates but transcribes denatured genomic N4 DNA or promoter-containing single-stranded DNA (ssDNA) with in vivo specificity (2, 3). vRNAP promoters contain a 5-to 7-bp stem, 3-nt loop hairpin, and conserved sequences (3, 4).In vivo, N4 early transcription requires Escherichia coli DNA gyrase and E. coli ssDNA-binding protein (EcoSSB), which provide an ''active'' promoter structure (5). Other SSBs (T7 gp 2.5, T4 gp 32, and N4 SSB) fail to stimulate vRNAP transcription in vitro because they destabilize the promoter hairpin (6). vRNAP transcripts generated from ssDNA templates are retained as RNA⅐DNA hybrids, suggesting that vRNAP cannot displace the RNA product (2). We show that EcoSSB activates vRNAP transcription on ssDNA templates through displacement of the RNA product and, therefore, through template recycling.The 177-aa EcoSSB polypeptide consists of a 115-aa Nterminal domain responsible for DNA binding and an Ϸ50-aa Pro-and Gly-rich sequence followed by a 10-aa acidic tail, highly conserved in eubacterial SSBs (7,8). Human mitochondrial (Hsmt) SSB is similar in sequence and structure to the Nterminal domain of EcoSSB but lacks the conserved C-terminal sequence (9-11). We show that Hsmt SSB did not activate vRNAP transcription although it did not disrupt the promoter hairpin. We determined that the 10 C-terminal ami...

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“…Further regulatory work with these and other mutants of topoisomerases is also needed to elucidate the molecular mechanism involved in the DNA supercoiling-mediated modulation of dnaQ expression. An alternative, but more improbable, hypothesis might be the assumption that the mutant SSB-113 protein stimulates the transcription from the dnaQP2 promoter in a similar way to that described for the transcriptional activation of the N4 virion RNA polymerase (Markiewicz et al, 1992). In that work, SSB has been characterized as a supercoiled template-dependent transcriptional activator.…”

Section: Discussionmentioning

confidence: 99%

The ssb‐113 allele suppresses the dnaQ49 mutator and alters DNA supercoiling in Escherichia coli

Quiñones

Neumann

1997

Molecular Microbiology

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SummaryMutations in the dnaQ gene, which encodes the proofreading ⑀-subunit of the DNA polymerase III holoenzyme, lead to a mutator phenotype caused by enhanced error rates during DNA replication. In this paper, we studied the influence of ssb mutations on the dnaQ49 mutator, because of the involvement of SSB protein in DNA replication. We found that the ssb-113 mutation suppresses the mutator phenotype of dnaQ49. The suppression effect resulted from an enhanced expression of the dnaQ49 allele as determined by experiments with gene fusions. S1 nuclease analysis revealed that the increased dnaQ expression is based on transcriptional activation of the dnaQP2 promoter. This seems to be the consequence of an increased DNA supercoiling in the ssb-113 mutant, which also influenced further functions that are sensitive to alterations in DNA supercoiling. These results support the hypothesis that the expression of the ⑀-subunit of DNA polymerase III may additionally be modulated by DNA supercoiling, and suggest a possible role for DNA topology in mutagenesis.

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Escherichia coli single-stranded DNA-binding protein is a supercoiled template-dependent transcriptional activator of N4 virion RNA polymerase.

Cited by 38 publications

References 69 publications

Supercoil-induced extrusion of a regulatory DNA hairpin

Supercoil-induced extrusion of a regulatory DNA hairpin

Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase

The ssb‐113 allele suppresses the dnaQ49 mutator and alters DNA supercoiling in Escherichia coli

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