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

Abstract

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...

The bacteriophage T4 AsiA protein contacts the β-flap domain of RNA polymerase

“…We therefore wanted to identify amino acid substitutions within the flap-tip helix that weakened the AsiA/␤-flap interaction, but did not prevent recognition of Ϫ10/Ϫ35 promoters. We tested the effects of several previously characterized amino acid substitutions in the ␤-flap-tip helix (31) and found that substitution G907K moderately disrupted the AsiA/␤-flap interaction, and amino acid substitutions I905A and F906A in combination strongly disrupted the AsiA/␤-flap interaction (Fig. 3 E and F).…”

“…Prior work has established that the ␤-flap-tip helix (␤ residues 900-909) is the primary determinant of the 70 region 4/␤-flap interaction (30) and that removal of the ␤-flap-tip helix eliminates any detectable interaction between 70 region 4 and the ␤-flap in the 2-hybrid assay (31). We found that removal of the ␤-flap-tip helix also eliminated the interaction between AsiA and the ␤-flap ( Fig.…”

“…S3 and ref. 31), reconstituted RNAP holoenzymes bearing these substitutions can initiate transcription from Ϫ10/Ϫ35 promoters in vitro (31).…”

Distinct pathways of RNA polymerase regulation by a phage-encoded factor

“…The antitermination activity of N and Q, but not gp39, is enhanced by cell-encoded proteins, including transcription elongation factor NusA, which by itself stimulates termination but reverses its activity to additionally stabilize the TEC in cooperation with N and Q (5, 6). Curiously, the N, Q, gp39, and NusA proteins all target the flexible β flap domain of RNAP (2,4,(7)(8)(9)(10)(11) that forms a part of the RNA exit channel and has been implicated in intrinsic termination and transcription pausing through direct interaction with RNA hairpins (Fig. 1) (3,(12)(13)(14).…”

Transcription elongation

“…56 Q becomes a subunit of RNA polymerase at the s70-dependent pause site at C16 described above, where a DNA binding site for Q is exposed. 66,67 The primary activity of Q relevant to the mechanism of antitermination is that binding of Q to the paused RNAP drives it out of the pause; 68,69 thus, the Q modification must counteract the pausing element, which we know is an EPS. Since an EPS also likely underlies the function of terminators, such antipausing may constitute the essential act of antitermination.…”