Structural basis for transcription elongation by bacterial RNA polymerase

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...

Section: Discussionsupporting

confidence: 62%

mentioning

confidence: 99%

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

Deighan

Diez²,

Leibman³

et al. 2008

“…A structurally conserved element, the trigger loop, has been suggested to play a key role in both selection of the correctly matched NTP and catalysis of transcription elongation in eukaryotes and prokaryotes (8)(9)(10). Structures of the transcribing complex reported by Wang et al, (8) reveal the trigger loop in a previously unseen "closed" conformation, making a network of interactions with the correctly matched NTP in the A site.…”

mentioning

confidence: 99%

RNA polymerase II trigger loop residues stabilize and position the incoming nucleotide triphosphate in transcription

Huang

Wang

Weiss

et al. 2010

109

A structurally conserved element, the trigger loop, has been suggested to play a key role in substrate selection and catalysis of RNA polymerase II (pol II) transcription elongation. Recently resolved X-ray structures showed that the trigger loop forms direct interactions with the β-phosphate and base of the matched nucleotide triphosphate (NTP) through residues His1085 and Leu1081, respectively. In order to understand the role of these two critical residues in stabilizing active site conformation in the dynamic complex, we performed all-atom molecular dynamics simulations of the wildtype pol II elongation complex and its mutants in explicit solvent. In the wild-type complex, we found that the trigger loop is stabilized in the "closed" conformation, and His1085 forms a stable interaction with the NTP. Simulations of point mutations of His1085 are shown to affect this interaction; simulations of alternative protonation states, which are inaccessible through experiment, indicate that only the protonated form is able to stabilize the His1085-NTP interaction. Another trigger loop residue, Leu1081, stabilizes the incoming nucleotide position through interaction with the nucleotide base. Our simulations of this Leu mutant suggest a three-component mechanism for correctly positioning the incoming NTP in which (i) hydrophobic contact through Leu1081, (ii) base stacking, and (iii) base pairing work together to minimize the motion of the incoming NTP base. These results complement experimental observations and provide insight into the role of the trigger loop on transcription fidelity. nucleotide selection | transcription fidelity

“…1). In the absence of a nucleotide substrate, the TL [amino acids 1234-1254 in Thermus aquaticus (Taq) and Thermus thermophilus RNAPs; amino acids 1077-1097 in Saccharomyces cerevisiae (Sce) RNAPII] adopts an open conformation in which its central part is unstructured (1,2). Binding of an NTP substrate induces folding of the TL, resulting in extension of the two ␣-helixes at the base of the TL and creating a closed, catalytically competent conformation of the active center in which the NTP is properly aligned with the 3Ј-OH of the nascent RNA for facile catalysis ( Fig.…”

mentioning

confidence: 99%

Allosteric control of catalysis by the F loop of RNA polymerase

Miropolskaya

Artsimovitch

Klimašauskas³

et al. 2009

Self Cite

Bacterial RNA polymerases (RNAPs) undergo coordinated conformational changes during catalysis. In particular, concerted folding of the trigger loop and rearrangements of the bridge helix at the RNAP active center have been implicated in nucleotide addition and RNAP translocation. At moderate temperatures, the rate of catalysis by RNAP from thermophilic Thermus aquaticus is dramatically reduced compared with its closest mesophilic relative, Deinococcus radiodurans. Here, we show that a part of this difference is conferred by a third element, the F loop, which is adjacent to the N terminus of the bridge helix and directly contacts the folded trigger loop. Substitutions of amino acid residues in the F loop and in an adjacent segment of the bridge helix in T. aquaticus RNAP for their D. radiodurans counterparts significantly increased the rate of catalysis (up to 40-fold at 20°C). A deletion in the F loop dramatically impaired the rate of nucleotide addition and pyrophosphorolysis, but it had only a moderate effect on intrinsic RNA cleavage. Streptolydigin, an antibiotic that blocks folding of the trigger loop, did not inhibit nucleotide addition by the mutant enzyme. The resistance to streptolydigin likely results from the loss of its functional target, the folding of the trigger loop, which is already impaired by the F-loop deletion. Our results demonstrate that the F loop is essential for proper folding of the trigger loop during nucleotide addition and governs the temperature adaptivity of RNAPs in different bacteria.nucleotide addition ͉ RNA cleavage ͉ streptolydigin ͉ temperature adaptation ͉ transcription C ellular multisubunit RNA polymerases (RNAPs) transcribe genes of up to tens of thousands nucleotides long with high precision and processivity. The catalytic cycle of RNAP is driven by complex conformational changes that accompany NTP binding, catalysis, and RNAP translocation. Recent studies identified two elements in the largest RNAP subunit (␤Ј in bacteria, Rpb1 in eukaryotic RNAP), the trigger loop (TL, or G loop), and the bridge helix (BH, or F-bridge helix), which appear to play the key role during the nucleotide addition cycle (Fig. 1). In the absence of a nucleotide substrate, the TL [amino acids 1234-1254 in Thermus aquaticus (Taq) and Thermus thermophilus RNAPs; amino acids 1077-1097 in Saccharomyces cerevisiae (Sce) RNAPII] adopts an open conformation in which its central part is unstructured (1, 2). Binding of an NTP substrate induces folding of the TL, resulting in extension of the two ␣-helixes at the base of the TL and creating a closed, catalytically competent conformation of the active center in which the NTP is properly aligned with the 3Ј-OH of the nascent RNA for facile catalysis (Fig. 1 A-C) (3, 4). The folded TL and the BH form a three-helix bundle that interacts with the substrate NTP and the template DNA base. In particular, the TL residues M1238 and H1242 (Taq numbering is used throughout the manuscript unless otherwise indicated) contact the base and the phosphate moieties of the ...