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

Davydova, Elena K.; Rothman-Denes, Lucia B.

doi:10.1073/pnas.1133325100

Cited by 23 publications

(26 citation statements)

References 51 publications

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“…The 100% cross-linked material was excised and eluted from the gel. Runoff transcription reactions were performed for 5 min at 37°C in the presence of 10 ⌴ E. coli SSB and analyzed as described previously (4).…”

Section: Cross-linked Stem Hairpin Template For Promotermentioning

confidence: 99%

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Bacteriophage N4 virion RNA polymerase interaction with its promoter DNA hairpin

Davydova

Santangelo²,

Rothman-Denes³

2007

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

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Bacteriophage N4 minivirion RNA polymerase (mini-vRNAP), the RNA polymerase (RNAP) domain of vRNAP, is a member of the T7-like RNAP family. Mini-vRNAP recognizes promoters that comprise conserved sequences and a 3-base loop-5-base pair (bp) stem DNA hairpin structure on single-stranded templates. Here, we defined the DNA structural and sequence requirements for minivRNAP promoter recognition. Mini-vRNAP binds a 20-nucleotide (nt) N4 P2 promoter deoxyoligonucleotide with high affinity (K d ‫؍‬ 2 nM) to form a salt-resistant complex. We show that mini-vRNAP interacts specifically with the central base of the hairpin loop (؊11G) and a base at the stem (؊8G) and that the guanine 6-keto and 7-imino groups at both positions are essential for binding and complex salt resistance. The major determinant (؊11G), which must be presented to mini-vRNAP in the context of a hairpin loop, appears to interact with mini-vRNAP Trp-129. This interaction requires template single-strandedness at positions ؊2 and ؊1. Contacts with the promoter are disrupted when the RNA product becomes 11-12 nt long. This detailed description of vRNAP interaction with its promoter hairpin provides insights into RNAPpromoter interactions and explains how the injected vRNAP, which is present in one or two copies, recognizes its promoters on a single copy of the injected genome.T7-like RNA polymerase B acteriophage N4 virion RNA polymerase (vRNAP), which is responsible for the transcription of the phage early genes, is present in virions in one or two copies and is injected into the host together with the phage genome at the onset of infection (1, 2). vRNAP is a 3,500-aa polypeptide that lacks extensive sequence similarity to either of the two known families of DNA-dependent RNAPs (3). Using controlled trypsin proteolysis and catalytic autolabeling, we defined a stable and transcriptionally active 1,106-aa domain (mini-vRNAP) located at the center of the vR-NAP polypeptide (3). Mini-vRNAP possesses the same initiation, elongation, termination, and product displacement properties as full-length vRNAP (3, 4).Mutational, biochemical, and phylogenetic analyses indicated that mini-vRNAP is a highly diverged member of the T7 RNAP family (3). However, although T7 RNAP recognizes its promoters in double-stranded templates (5), vRNAP transcribes promotercontaining, single-stranded templates with in vivo specificity (6), recognizing a 3-base loop-5-bp stem hairpin structure and specific sequences at the promoter (7). In vivo, vRNAP promoter recognition requires template supercoiling (8) to drive extrusion of the promoter hairpins from the double-stranded N4 genome (9, 10) and Escherichia coli single-stranded DNA-binding protein to stabilize single-stranded regions surrounding the site of transcription initiation (11). We have used single-stranded templates to identify unambiguously the site of transcription initiation as well as the sequence and structural DNA determinants of vRNAP-promoter interaction. The results reveal two major determinants of vRNAP hairpin rec...

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Section: Cross-linked Stem Hairpin Template For Promotermentioning

confidence: 99%

“…Using controlled trypsin proteolysis and catalytic autolabeling, we defined a stable and transcriptionally active 1,106-aa domain (mini-vRNAP) located at the center of the vR-NAP polypeptide (3). Mini-vRNAP possesses the same initiation, elongation, termination, and product displacement properties as full-length vRNAP (3,4).…”

mentioning

confidence: 99%

Bacteriophage N4 virion RNA polymerase interaction with its promoter DNA hairpin

Davydova

Santangelo²,

Rothman-Denes³

2007

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

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“…Whether the N-terminal domain undergoes a structural rearrangement upon proceeding from initiation to transcript elongation, which enlarges the pore sufficiently to enable the A-form DNA-RNA hybrid to exit from the complex without strand separation, remains to be determined. EcoSSB activates vRNAP transcription at limiting single-stranded template concentrations through template recycling, a function that has been mapped to the C-terminal 10 residues of the EcoSSB (21). We have proposed that EcoSSB functionally substitutes for part of the N-terminal domain of RNAP that should be responsible for RNA separation from template DNA during N4 vRNAP elongation (21).…”

Section: Resultsmentioning

confidence: 99%

“…The N4 vRNAP RNA product is not displaced from template DNA, thus limiting template DNA usage to one round (21). Modeling of the DNA-RNA hybrid in the N4 minivRNAP structure (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The vRNAP recognizes promoters that are composed of a 5-bp stem (nucleotides Ϫ17 to Ϫ13 and Ϫ9 to Ϫ5) and a three base-loop (nucleotides Ϫ12 to Ϫ10) hairpin with conserved sequence (18,19). The Escherichia coli single-stranded DNA-binding protein EcoSSB is required for vRNAP transcription initiation and elongation; other single-stranded DNA-binding proteins cannot substitute (20,21).…”

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confidence: 99%

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X-ray crystal structure of the polymerase domain of the bacteriophage N4 virion RNA polymerase

Murakami

Davydova

Rothman-Denes

2008

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

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Coliphage N4 virion RNA polymerase (vRNAP), which is injected into the host upon infection, transcribes the phage early genes from promoters that have a 5-bp stem-3 nt loop hairpin structure. Here, we describe the 2.0-Å resolution x-ray crystal structure of N4 mini-vRNAP, a member of the T7-like, single-unit RNAP family and the minimal component having all RNAP functions of the fulllength vRNAP. The structure resembles a ''fisted right hand'' with Fingers, Palm and Thumb subdomains connected to an N-terminal domain. We established that the specificity loop extending from the Fingers along with W129 of the N-terminal domain play critical roles in hairpin-promoter recognition. A comparison with the structure of the T7 RNAP initiation complex reveals that the pathway of the DNA to the active site is blocked in the apo-form vRNAP, indicating that vRNAP must undergo a large-scale conformational change upon promoter DNA binding and explaining the highly restricted promoter specificity of vRNAP that is essential for phage early transcription. RNA polymerases (RNAPs) belonging to the T7-like family consist of a single catalytic polypeptide of Ϸ100 kDa. This family includes phage-encoded, chloroplast and mitochondrial nuclear-encoded and linear plasmid-encoded enzymes (1). T7 RNAP has been the most extensively studied member of the family, and several crystal structures have been determined (2-5). However, with the exception of T7 and closely related phage T3, SP6, and K11 RNAPs, other members of the family require additional factors for transcription initiation. The yeast mitochondrial RNAP requires transcription factor Mtf1 (6, 7), which associates with the catalytic core RPO41 to form a holoenzyme that is competent for transcription initiation (8). It has been suggested that Mft1 plays a role in promoter melting, because yeast mitochondrial RNAP can initiate specifically on supercoiled or premelted (bubble) templates in the absence of Mtf1 (9). The human mitochondrial RNAP, POLRMT (10), accurately transcribes templates containing its cognate promoters only when supplemented with the high-mobility-group (HMG) protein TFAM (11, 12) along with either TFB1M or TFB2M transcription factors (13).The early region of the double-stranded linear DNA genome of lytic coliphage N4 is transcribed by a phage-coded, virionencapsidated RNAP (vRNAP), a 3,500-aa uncleaved polyprotein that is injected into the host cell with the phage genome at the onset of infection (14-16). Limited proteolysis experiments established that the N4 vRNAP polyprotein is composed of three functional domains; the central domain of Ϸ1,100 aa (minivRNAP), which possesses the same transcriptional specificity and properties as full-length vRNAP, is the most evolutionarily diverged member of the T7-like RNAP family (17). The vRNAP recognizes promoters that are composed of a 5-bp stem (nucleotides Ϫ17 to Ϫ13 and Ϫ9 to Ϫ5) and a three base-loop (nucleotides Ϫ12 to Ϫ10) hairpin with conserved sequence (18,19). The Escherichia coli single-stranded DNA-binding protein ...

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Functions of Single-Strand DNA-Binding Proteins in DNA Replication, Recombination, and Repair

Marceau

2012

Methods in Molecular Biology

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Double-stranded (ds) DNA contains all of the necessary genetic information, although practical use of this information requires unwinding of the duplex DNA. DNA unwinding creates single-stranded (ss) DNA intermediates that serve as templates for myriad cellular functions. Exposure of ssDNA presents several problems to the cell. First, ssDNA is thermodynamically less stable than dsDNA, which leads to spontaneous formation of duplex secondary structures that impede genome maintenance processes. Second, relative to dsDNA, ssDNA is hypersensitive to chemical and nucleolytic attacks that can cause damage to the genome. Cells deal with these potential problems by encoding specialized ssDNA-binding proteins (SSBs) that bind to and stabilize ssDNA structures required for essential genomic processes. SSBs are essential proteins found in all domains of life. SSBs bind ssDNA with high affinity and in a sequence-independent manner and, in doing so, SSBs help to form the central nucleoprotein complex substrate for DNA replication, recombination, and repair processes. While SSBs are found in every organism, the proteins themselves share surprisingly little sequence similarity, subunit composition, and oligomerization states. All SSB proteins contain at least one DNA-binding oligonucleotide/oligosaccharide binding (OB) fold, which consists minimally of a five stranded beta-sheet arranged as a beta barrel capped by a single alpha helix. The OB fold is responsible for both ssDNA binding and oligomerization (for SSBs that operate as oligomers). The overall organization of OB folds varies between bacteria, eukaryotes, and archaea. As part of SSB/ssDNA cellular structures, SSBs play direct roles in the DNA replication, recombination, and repair. In many cases, SSBs have been found to form specific complexes with diverse genome maintenance proteins, often helping to recruit SSB/ssDNA-processing enzymes to the proper cellular sites of action. This clustering of genome maintenance factors can help to stimulate and coordinate the activities of individual enzymes and is also important for dislodging SSB from ssDNA. These features support a model in which DNA metabolic processes have evolved to work on ssDNA/SSB nucleoprotein filaments rather than on naked ssDNA. In this volume, methods are described to interrogate SSB-DNA and SSB-protein binding functions along with approaches that aim to understand the cellular functions of SSB. This introductory chapter offers a general overview of SSBs that focuses on their structures, DNA-binding mechanisms, and protein-binding partners.

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

Cited by 23 publications

References 51 publications

Bacteriophage N4 virion RNA polymerase interaction with its promoter DNA hairpin

Bacteriophage N4 virion RNA polymerase interaction with its promoter DNA hairpin

X-ray crystal structure of the polymerase domain of the bacteriophage N4 virion RNA polymerase

Functions of Single-Strand DNA-Binding Proteins in DNA Replication, Recombination, and Repair

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