Active site opening and closure control translocation of multisubunit RNA polymerase

Abstract

Multisubunit RNA polymerase (RNAP) is the central information-processing enzyme in all cellular life forms, yet its mechanism of translocation along the DNA molecule remains conjectural. Here, we report direct monitoring of bacterial RNAP translocation following the addition of a single nucleotide. Time-resolved measurements demonstrated that translocation is delayed relative to nucleotide incorporation and occurs shortly after or concurrently with pyrophosphate release. An investigation of translocation equilibrium suggested that the strength of interactions between RNA 3' nucleotide and nucleophilic and substrate sites determines the translocation state of transcription elongation complexes, whereas active site opening and closure modulate the affinity of the substrate site, thereby favoring the post- and pre-translocated states, respectively. The RNAP translocation mechanism is exploited by the antibiotic tagetitoxin, which mimics pyrophosphate and induces backward translocation by closing the active site.

Figure 1.

An overview of bacterial TEC structure. β (light blue), β′ (wheat) and two (gray) subunits are depicted as transparent surfaces; the non-essential ω subunit is obstructed by β and β′ subunits. Template DNA, non-template DNA and RNA are depicted in black, green and red, respectively. The β′ subunit elements discussed throughout the manuscript are depicted as cartoons: the folded TL is colored cyan; the bridge helix (BH), lid loop, TL base and catalytic aspartate triad loop are colored orange. The β-pincer domain is outlined. Substrate NTP atoms are shown as spheres. RNAP is drawn using coordinates of Thermus thermophilus TEC PDB ID 2O5J, and nucleic acids are taken from the E. coli TEC model (15).

Figure 2.

Overview of the experimental setup used in this study. RNAP is shaded gray. Two alternative conformations of the TL domain are indicated, with the predominant conformation shaded in turquoise (see ‘Discussion’ section). Template DNA, non-template DNA and RNA are depicted in black, green and red, respectively. Although presented as a bifluorophore for compact representation, each TEC in our experiments possessed only one of the indicated fluorophores: either guanine analog 6-MI (cyan) at i − 7 or adenine analog 2-AP (violet) at i + 2 (for the initial TECs). Guanine, serving as a quencher, is depicted in yellow.

Figure 3.

Kinetic and equilibrium analyses of translocation following CMP incorporation. Top: The initial TEC schematic. Left: Time-resolved measurements of RNA extension by a single CMP nucleotide (olive), release of the reaction by-product PPi (dark teal; simulated as described in Supplementary Figure S3), and translocation of the RNA–DNA hybrid within the RNAP main channel (red). Inset: Translocation curves measured over a 10-min interval in the absence (red) and presence (cyan) of 5 µM TGT. Center: Denaturing polyacrylamide gel of TEC RNAs and fluorescence levels of initial (gray), and CMP (red)-, 2′-dCMP (blue)- and 3′-dCMP (black)-extended TECs. Here and in all subsequent figures, fluorescence levels were normalized as described in Supplementary Methods. Note the faster electrophoretic mobility of 2′- and 3′-dCMP-extended RNAs. Right: Titration of extended TECs with TGT.

Figure 4.

Contributions of the individual steps to the overall transcription rate. The half-lives of nucleotide addition, PP_i release and translocation reactions are depicted as olive, turquoise and red bars, respectively. Purple bars depict the combined half-life of PP_i hydrolysis into P_i and P_i binding to MDCC-PBP (Supplementary Figure S3), which accounted for the 5.8-ms delay in the fluorescence signal in PP_i release measurements. Error bars are SDs of two to three independent experiments. AMP, CMP, GMP and UMP data were obtained with i − 7 6-MI TECs; AMP* data were obtained with i + 2 2-AP TEC (Supplementary Figure S2).

Figure 5.

The effects of amino acid substitutions in RNAP on translocation equilibrium. Schematics of the initial scaffolds are indicated above the graphs. (A) TGT titration of AMP-extended TECs assembled using wild-type (red), ΔTL (blue), β′N458D (green), β'R933A (orange), β′H936A (cyan) and β′M932A (black) RNAPs. (B) TGT titration of 2-AP ribonucleoside monophosphate-extended TECs assembled using wild-type and β′M932A RNAPs. Inset: Upon TL folding (dark teal helix) and backward translocation, the sulfur atom of β′Met932 (yellow sphere) closely (∼5 Å) approaches the 2-AP nucleobase (violet) at the RNA 3′ end, thereby quenching its fluorescence. Active-site Mg²⁺ is depicted as a magenta sphere. Catalytic aspartates and β′Asn458 are shown as sticks. The representation is drawn using the model of the pre-translocated TEC of T. thermophilus RNAP (see Supplementary Figure S4A); residue numbers correspond to E. coli RNAP.

Figure 6.

Normalized fluorescence of GMP-extended TEC as a function of TGT and CMPcPP concentrations. A schematic of the initial scaffold and kinetic model used for global analysis of titration data is indicated above the graph. Reaction species in the model are abbreviated as follows: TEC_post, post-translocated TEC17; TEC_preTGT, pre-translocated TEC17 with bound TGT; TEC_postCMPcPP, post-translocated TEC17 with bound CMPcPP. CMPcPP and TGT dissociation constants are indicated. The best-fit value of the TGT dissociation constant is 2-fold higher than the value for GMP-extended TEC reported in Table 1 due to the use of a different batch of TGT.

Figure 7.

RNA–protein contacts (green dashed lines) specific for the post- and pre-translocated states. The active site Mg²⁺ ion is depicted as a magenta sphere. Residue numbers correspond to E. coli RNAP. (A) The post-translocated state (PDB 2O5I). (B) The pre-translocated state model, generated as described in Supplementary Figure S4A. (C) The effects of Mg²⁺ withdrawal on translocation equilibrium in TECs extended with AMP, 2′ dAMP and 3′ dAMP. A schematic of the initial scaffold is indicated above the graph. The initial concentration of Mg²⁺ in the assay medium was 2 mM.

Figure 8.

Structural model of the closed TEC–TGT complex. Phosphate and carboxylate groups of TGT (brown) overlap with the PP_i molecule (yellow). TL is colored turquoise and the rest of RNAP is colored gray. RNA is colored rose. The surface contributed by TL residues for interaction with RNA 3′ NMP is contoured by a turquoise line. Asp triad residues and selected side chains that interact with TGT and the 3′ RNA nucleobase are shown as sticks. Active site Mg²⁺ ions coordinated by the Asp triad and PP_i are depicted as magenta and yellow spheres, respectively. TGT–protein contacts are indicated as green dashed lines.