T7 phage protein Gp2 inhibits the
            <i>Escherichia coli</i>
            RNA polymerase by antagonizing stable DNA strand separation near the transcription start site

Cámara, Beatríz; Liu, Minhao; Reynolds, Jonathan; Shadrin, Andrey M.; Liu, Bing; Kwok, King; Simpson, Peter; Weinzierl, Robert O. J.; Severinov, Konstantin; Cota, Ernesto; Matthews, Stephen; Wigneshweraraj, Sivaramesh

doi:10.1073/pnas.0907908107

Cited by 63 publications

(116 citation statements)

References 26 publications

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“…S1) intermediates on the pathway to the final R-loop complex. Similar methodology has been applied to study mechanisms of duplex DNA destabilization by RNA polymerases and a variety of enzymes that use base flipping to gain access to bases in the double-stranded DNA (17,18). To determine equilibrium dissociation constant (K d ) values of dCas9/sgRNA complexes with various probes quantitatively, we measured the ability of these probes to affect the rate of dCas9/sgRNA binding to "Cas9 beacons," fluorescently labeled DNA fragments containing the protospacer and PAM, competitively (19).…”

Section: Resultsmentioning

confidence: 99%

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

Mekler

Minakhin

Severinov

2017

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

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The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves doublestranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/ single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/ guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.Cas9 | CRISPR | DNA interrogation | protein-DNA interactions | fluorescence spectroscopy P rokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) gene systems acquire fragments of foreign DNA (protospacers) and insert them as spacers into the CRISPR array in the prokaryotic host genome. Upon subsequent encounters, the complex of Cas proteins with CRISPR RNA (crRNA) bearing the spacer sequence binds and cleaves foreign DNA containing the matching sequence (1-3), thus providing the host organism with adaptive hereditable immunity. The DNA endonuclease Cas9 of type II CRISPR-Cas systems is targeted to specific DNA sequences by a 20-base crRNA spacer that binds to the complementary strand of protospacer DNA, displacing the noncomplementary strand to form an R-loop (4, 5). Both binding and cleavage of target DNA by the Cas9/crRNA complex require a short protospacer adjacent motif (PAM) located immediately downstream of the targeted sequence (6). For the most commonly used Streptococcus pyogenes Cas9 (SpCas9), the PAM sequence is 5′-NGG (7). The perfect match between guide RNA and seven to 12 bases of target DNA at the immediate 5′ side of PAM ("seed" region) is the most critical for DNA binding and cleavage, although limited mispairing in the distal bases is tolerated (8). Two RNA molecules (crRNA and a transactivating crRNA (tracrRNA)) required to guide SpCas9 to targets can be replaced with...

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Section: Resultsmentioning

confidence: 99%

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

Mekler

Minakhin

Severinov

2017

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

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“…By binding to the β′-jaw inside the RNAP active site channel, Gp2 sterically (Fig. S5) and electrostatically (6,7) interferes with the proper positioning of DNA in the RNAP downstream duplex DNA channel. However, in the absence of σ 70 1:1 , promoter DNA can effectively compete with Gp2, so Gp2 inhibition is compromised (5-7).…”

Section: Resultsmentioning

confidence: 99%

“…S4) (19). The Gp2-β′ R56-E1188 and R58-E1158 interactions are critical for Gp2 binding to, and inhibition of, RNAP (4,6).…”

Section: Significancementioning

confidence: 99%

Phage T7 Gp2 inhibition of Escherichia coli RNA polymerase involves misappropriation of σ ⁷⁰ domain 1.1

Bae

Davis

Brown

et al. 2013

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

151

165

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Significance After infection of Escherichia coli by bacteriophage T7, the host RNA polymerase (RNAP) produces early phage transcription products that encode the phages own RNAP (that transcribes subsequent phage genes) as well as Gp2, an essential inhibitor of the host RNAP. X-ray crystal structures of E. coli RNAP define the structure and location of the RNAP σ 70 subunit domain 1.1 inside the RNAP active site channel, where it must be displaced by the DNA upon formation of the transcription complex. Gp2 binds inside the RNAP active site channel and also interacts with , preventing from exiting the RNAP active site channel. Gp2 thus misappropriates a domain of the RNAP, , to inhibit the function of the enzyme.

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“…Anti-s factors bind to a s factor and block its interaction with RNAP to prevent complete holoenzyme formation (Campbell et al 2003;Lambert et al 2004;Sorenson et al 2004;Baxter et al 2006). Some anti-s factors also interfere with the interaction between s and DNA by blocking the DNAbinding site of s. The T7 gp2 protein binds and occludes the DNA-binding channel of RNAP (Camara et al 2010;Nechaev and Severinov 2003). In contrast, the present study revealed that the phage protein gp39 functions via a novel, unanticipated mechanism by repositioning the promoter-binding determinant (s 4 ) without interacting with DNA, dissociating the host s factor from RNAP, masking the DNA-binding determinants of s, or occluding the RNAP channels.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for promoter specificity switching of RNA polymerase by a phage factor

Tagami

Sekine

Minakhin³

et al. 2014

Genes Dev.

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Transcription of DNA to RNA by DNA-dependent RNA polymerase (RNAP) is the first step of gene expression and a major regulation point. Bacteriophages hijack their host's transcription machinery and direct it to serve their needs. The gp39 protein encoded by Thermus thermophilus phage P23-45 binds the host's RNAP and inhibits transcription initiation from its major ''-10/-35'' class promoters. Phage promoters belonging to the minor ''extended -10'' class are minimally inhibited. We report the crystal structure of the T. thermophilus RNAP holoenzyme complexed with gp39, which explains the mechanism for RNAP promoter specificity switching. gp39 simultaneously binds to the RNAP b-flap domain and the C-terminal domain of the s subunit (region 4 of the s subunit [s 4 ]), thus relocating the b-flap tip and s 4 . The~45 Å displacement of s 4 is incompatible with its binding to the -35 promoter consensus element, thus accounting for the inhibition of transcription from -10/-35 class promoters. In contrast, this conformational change is compatible with the recognition of extended -10 class promoters. These results provide the structural bases for the conformational modulation of the host's RNAP promoter specificity to switch gene expression toward supporting phage development for gp39 and, potentially, other phage proteins, such as T4 AsiA.

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T7 phage protein Gp2 inhibits the Escherichia coli RNA polymerase by antagonizing stable DNA strand separation near the transcription start site

Cited by 63 publications

References 26 publications

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

Phage T7 Gp2 inhibition of Escherichia coli RNA polymerase involves misappropriation of σ ⁷⁰ domain 1.1

Structural basis for promoter specificity switching of RNA polymerase by a phage factor

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T7 phage protein Gp2 inhibits the Escherichia coli RNA polymerase by antagonizing stable DNA strand separation near the transcription start site

Cited by 63 publications

References 26 publications

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

Phage T7 Gp2 inhibition of Escherichia coli RNA polymerase involves misappropriation of σ 70 domain 1.1

Structural basis for promoter specificity switching of RNA polymerase by a phage factor

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Phage T7 Gp2 inhibition of Escherichia coli RNA polymerase involves misappropriation of σ ⁷⁰ domain 1.1