Inactive complex formation between E. coli RNA polymerase and inhibitor protein purified from T7 phage infected cells

Hesselbach, Bruce A.; Nakada, Dai

doi:10.1038/258354a0

Cited by 25 publications

(21 citation statements)

References 16 publications

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“…Early T7 genes are transcribed from several very strong promoters by E. coli RNAP containing primary σ 70 subunit (Eσ 70 ), whereas middle and late T7 genes are transcribed by phage-encoded RNAP 3 . The product of the middle T7 gene 2, a 7 kDa protein called gp2, binds to the β′ jaw domain of the host RNAP and inhibits it 4,5 . Only one gp2 molecule participates in tight interaction with an RNAP molecule 6 and point mutations in as well as deletions of the β′ jaw abolish this binding 5 .…”

Section: Introductionmentioning

confidence: 99%

Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein

Mekler

Minakhin

Sheppard

et al. 2011

Journal of Molecular Biology

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E. coli T7 bacteriophage gp2 protein is a potent inhibitor of host RNA polymerase (RNAP). Gp2 inhibits formation of open promoter complex by binding to the β′ jaw, an RNAP domain that interacts with downstream promoter DNA. Here, we used an engineered promoter with an optimized sequence to obtain and characterize a specific promoter complex containing RNAP and gp2. In this complex, localized melting of promoter DNA is initiated but does not propagate to include the point of the transcription start. As a result, the complex is transcriptionally inactive. Using a highly sensitive RNAP beacon assay we performed quantitative real-time measurements of specific binding of the RNAP-gp2 complex to promoter DNA and various promoter fragments. In this way, the effect of gp2 on RNAP interaction with promoters was dissected. As expected, gp2 greatly decreased RNAP affinity to downstream promoter duplex. However, gp2 also inhibited RNAP binding to promoter fragments that lacked downstream promoter DNA that interacts with the β′ jaw. The inhibition was caused by gp2-mediated decrease of the RNAP binding affinity to template and non-template strand segments of the transcription bubble downstream of the −10 promoter element. The inhibition of RNAP interactions with single-stranded segments of the transcription bubble by gp2 is a novel effect, which may occur via allosteric mechanism that is set in motion by the gp2 binding to the β′ jaw.

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Section: Introductionmentioning

confidence: 99%

Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein

Mekler

Minakhin

Sheppard

et al. 2011

Journal of Molecular Biology

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“…The requirement to shut off E. coli RNAP transcription is stringent. Gp2 is accordingly a potent inhibitor of E-σ 70 transcription initiation (3,4). Gp2 binds to E-σ 70 with sub-nanomolar K d (5).…”

Section: -Holoenzyme (E-σ 70mentioning

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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“…T7 bacteriophage gp2 protein inhibits RP o formation by binding to the RNAP downstream jaw domain and interfering with RNAP interactions with downstream promoter DNA (21,31). Another well studied inhibitor of open complex formation, T4 bacteriophage AsiA, binds to 70 region 4 and inhibits transcription from Ϫ35 element-dependent pro- Cys-TMR) 70 holoenzyme beacon with downstream fork junction probes.…”

Section: Rnap Binding To a Downstream Fork Junction Mimics Rnap Intermentioning

confidence: 99%

A Critical Role of Downstream RNA Polymerase-Promoter Interactions in the Formation of Initiation Complex

Mekler

Minakhin

Severinov

2011

Journal of Biological Chemistry

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Formation of the transcription-competent open promoter complex (RP o ) by DNA-dependent RNA polymerase (RNAP) 3 is a critical checkpoint on the pathway of gene expression. In bacteria, the transition from initial recognition of the promoter by RNAP to RP o proceeds through a series of short-lived intermediate complexes (1-3). In RP o , the DNA duplex is disrupted over a stretch of 12-15 base pairs, which leads to the formation of the transcription bubble and makes the transcription start point (position ϩ1) accessible to the RNAP catalytic center (4, 5). An important source of energy driving this strand separation process is the interaction of the RNAP 70 subunit region 2 with the non-template strand of the Ϫ10 promoter element at the upstream edge of the transcription bubble (6 -9). The interaction with the Ϫ10 element adenine at position Ϫ11 of the nontemplate strand is of special importance for nucleation of promoter melting (6, 10). RNAP interaction with the template segment of the transcription bubble was estimated to be considerably weaker than that with the non-template segment (8). Although formation of intermediates on the pathway to RP o has been characterized kinetically in many studies (1-3, 11, 12), it is still unclear which energetically favorable RNAP-promoter interactions are coupled with downstream propagation of the transcription bubble and determine its final boundary position at ϩ2/ϩ3.The crystal structures of prokaryotic RNAP indicate that establishment of RNAP interactions with the downstream duplex upon RP o formation must introduce a sharp kink in DNA, which may facilitate melting and stabilize the open complex (13,14). Indeed, RNAP mutations that change RNAP contacts with downstream DNA reduce the longevity of open complexes formed at certain promoters (15, 16). On highly supercoiled DNA, the downstream interactions may not be obligatory, as an RNAP subassembly containing an N-terminal fragment of the Escherichia coli RNAP ␤Ј subunit (lacking ␤Ј amino acids involved in contacts with downstream DNA) and a fragment of 70 subunit was reported to recognize and melt an extended Ϫ10 consensus promoter (17). However, melting observed in these experiments might not necessarily mirror steps in RP o formation by RNAP (3, 18). Downstream RNAPpromoter contacts are targeted by low molecular weight inhibitors and protein repressors that affect transcription initiation (16, 19 -21).Unraveling the role of downstream RNAP-promoter interactions in open complex formation and regulation of transcription initiation is hindered by the lack of experimental tools to directly measure their strength and specificity. Upon formation of the open promoter complex, two DNA fork junction structures are created around positions Ϫ12 and ϩ2. Studies of RNAP interactions with model DNA fragments mimicking the upstream fork junction provided important structural (22) and functional insights into the processes of promoter recognition and melting (8,9,(23)(24)(25)(26). Here, we introduce downstream fork junction fragments as ...

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Inactive complex formation between E. coli RNA polymerase and inhibitor protein purified from T7 phage infected cells

Cited by 25 publications

References 16 publications

Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein

Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein

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

A Critical Role of Downstream RNA Polymerase-Promoter Interactions in the Formation of Initiation Complex

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Inactive complex formation between E. coli RNA polymerase and inhibitor protein purified from T7 phage infected cells

Cited by 25 publications

References 16 publications

Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein

Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein

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

A Critical Role of Downstream RNA Polymerase-Promoter Interactions in the Formation of Initiation Complex

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