A Hydrophobic Patch on the Flap-tip Helix of E.coli RNA Polymerase Mediates σ70 Region 4 Function

Geszvain, Kati; Gruber, Tanja M.; Mooney, Rachel A.; Gross, Carol A.; Landick, Robert

doi:10.1016/j.jmb.2004.08.063

Cited by 56 publications

(75 citation statements)

References 57 publications

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“…1 A), we introduced mutations in the B. subtilis ␤-flap that are known to affect interaction negatively between the E. coli ␤-flap (I905K and F906K; Fig. 1 A) and 70 -region 4 (21). As predicted, the corresponding B. subtilis flap mutations (I864K and F865K) dramatically reduced the observed interaction between region 4 of YvrI and the ␤-flap (Fig.…”

Section: Yvri Regionsupporting

confidence: 56%

“…Region 4.2 includes a helix-turn-helix (HTH) domain that recognizes and binds the -35 element. Region 4 also interacts with hydrophobic amino acids in the ␤-flap domain of the core RNAP ␤-subunit (21), and this interaction properly orients the HTH domain for interaction with -35 region DNA (22). Additionally, region 4 is a frequent target for regulators that modulate RNAP activity through interaction with (23).…”

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confidence: 99%

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A two-subunit bacterial σ-factor activates transcription in Bacillus subtilis

MacLellan

Guariglia-Oropeza

Gaballa

et al. 2009

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

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The -like factor YvrI and coregulator YvrHa activate transcription from a small set of conserved promoters in Bacillus subtilis. We report here that these two proteins independently contribute -region 2 and -region 4 functions to a holoenzyme-promoter DNA complex. YvrI binds RNA polymerase (RNAP) through a region 4 interaction with the ␤-subunit flap domain and mediates specific promoter recognition but cannot initiate DNA melting at the -10 promoter element. Conversely, YvrHa possesses sequence similarity to a conserved core-binding motif in -region 2 and binds to the N-terminal coiled-coil element in the RNAP ␤-subunit previously implicated in interaction with region 2 of -factors. YvrHa plays an essential role in stabilizing the open complex and interacts specifically with the N-terminus of YvrI. Based on these results, we propose that YvrHa is situated in the transcription complex proximal to the -10 element of the promoter, whereas YvrI is responsible for -35 region recognition. This system presents an unusual example of a two-subunit bacterial -factor.

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Section: Yvri Regionsupporting

confidence: 56%

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confidence: 99%

A two-subunit bacterial σ-factor activates transcription in Bacillus subtilis

MacLellan

Guariglia-Oropeza

Gaballa

et al. 2009

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

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“…To do this, we tested the effect of removing the flap-tip helix (␤ residues 900-909), the primary target of 70 region 4 (24). Removal of the flap-tip helix abolished the interaction between the ␤-flap and each of the Q proteins (Fig.…”

Section: Resultsmentioning

confidence: 99%

The bacteriophage λ Q antiterminator protein contacts the β-flap domain of RNA polymerase

Deighan

Diez²,

Leibman³

et al. 2008

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

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The multisubunit RNA polymerase (RNAP) in bacteria consists of a catalytically active core enzyme (␣ 2␤␤ ) complexed with a factor that is required for promoter-specific transcription initiation. During early elongation the stability of interactions between and core decreases, in part because of the nascent RNA-mediated destabilization of an interaction between region 4 of and the flap domain of the ␤-subunit (␤-flap). The nascent RNA-mediated destabilization of the region 4/␤-flap interaction is required for the bacteriophage Q antiterminator protein ( Q) to engage the RNAP holoenzyme. Here, we provide an explanation for this requirement by showing that Q establishes direct contact with the ␤-flap during the engagement process, thus competing with 70 region 4 for access to the ␤-flap. We also show that Q's affinity for the ␤-flap is calibrated to ensure that Q activity is restricted to the late promoter PR . Specifically, we find that strengthening the Q/␤-flap interaction allows Q to bypass the requirement for specific cis-acting sequence elements, a Q-DNA binding site and a RNAP pause-inducing element, that normally ensure Q is recruited exclusively to transcription complexes associated with P R . Our findings demonstrate that the ␤-flap can serve as a direct target for regulators of elongation.sigma ͉ transcription elongation T he bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytically active core enzyme (␣ 2 ␤␤Ј ) in complex with a factor, which confers on the core enzyme the ability to initiate promoter-specific transcription (reviewed in ref. 1). In the context of the RNAP holoenzyme, 70 (the primary factor in Escherichia coli) contacts the conserved Ϫ10 and Ϫ35 promoter elements. All primary factors share four regions of conserved sequence (regions 1-4) (2). Regions 2 and 4 contain DNA-binding domains responsible for recognition of the promoter Ϫ10 element and Ϫ35 element, respectively. Holoenzyme formation depends on an interaction between 70 region 2 and a coiled-coil motif in the ␤Ј-subunit (the ␤Ј coiled coil) (3, 4); this interaction is also required for 70 to make functional contact with the promoter Ϫ10 element (5). Interaction between 70 region 4 and the flap domain of the ␤-subunit (the ␤-flap), although not required for holoenzyme formation, is required for contact with the promoter Ϫ35 element (6). In particular, the 70 region 4/␤-flap interaction properly positions 70 region 4 with respect to 70 region 2, thus enabling 70 region 4 and 70 region 2 to simultaneously contact promoter elements separated by Ϸ17 bp (6).During early elongation the stability of interactions between 70 and core RNAP decreases (reviewed in ref. 7), in part because of the nascent RNA-mediated destabilization of the 70 region 4/␤-flap interaction (8). Specifically, 70 region 4, when bound to the ␤-flap, is positioned within the path of the nascent RNA as it first emerges from the RNA exit channel (the channel through which the nascent RNA is extruded during transcription elongation) (9, 10) at a nascent RN...

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“…These hydrophobic contacts are seen directly within the T. thermophilus RNAP structure (2) (Fig. 5B), and conserved hydrophobic residues within the ␤-flap tip of E. coli (Leu-901, Leu-902, Ile-905, and Phe-906) are important for growth, for transcription from a Ϫ35/Ϫ10 promoter, and for an interaction with 70 (31). A hydrophobic cleft within MotA NTD also serves as the contact site for H5 (9).…”

Section: Discussionmentioning

confidence: 99%

Bacteriophage T4 MotA Activator and the β-Flap Tip of RNA Polymerase Target the Same Set of σ70 Carboxyl-terminal Residues

Bonocora

Decker

Glass

et al. 2011

Journal of Biological Chemistry

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Background: Transcriptional activators interact with RNA polymerase to redefine gene expression. Results: A phage activator engages a region of the specificity factor of E. coli RNA polymerase, which is normally bound by another portion of RNA polymerase. Conclusion: Using an activator/co-activator system, the phage hijacks the host RNA polymerase. Significance: Small transcriptional factors acting on defined local regions of RNA polymerase can fundamentally change gene expression.

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A Hydrophobic Patch on the Flap-tip Helix of E.coli RNA Polymerase Mediates σ70 Region 4 Function

Cited by 56 publications

References 57 publications

A two-subunit bacterial σ-factor activates transcription in Bacillus subtilis

A two-subunit bacterial σ-factor activates transcription in Bacillus subtilis

The bacteriophage λ Q antiterminator protein contacts the β-flap domain of RNA polymerase

Bacteriophage T4 MotA Activator and the β-Flap Tip of RNA Polymerase Target the Same Set of σ70 Carboxyl-terminal Residues

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