Allosteric control of catalysis by the F loop of RNA polymerase

Abstract

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 degrees 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.

Fig. 1.

Structure of the RNAP active site and location of substitutions in the F loop. (A–C) Three different views of the active center in the EC of T. thermophilus RNAP with the incoming NTP substrate (black) in the insertion site (3). The whole EC structure is shown in A at the bottom; the α-, β-, β′-, and ω-subunits are light blue, light green, light magenta, and gray, respectively, and the enlarged part of the active center is boxed. Views in B and C are successively rotated relative to the view in A by 90° and 45°. Two catalytic Mg²⁺ ions are shown as red spheres, the template DNA (positions +2 to −9) is orange, RNA (positions −1 to −9) is yellow, the F loop is red, the BH is magenta, and the TL is green. Amino acid residues at the tip of the TL (1243–1247) are shown in turquoise as a CPK model; residues in the F loop that are close to the tip of TL are red. M1238 and H1242 in the TL are green. Amino acid substitutions in the F loop in Taq/Dra RNAPs are shown in light yellow, Q1046 is dark yellow, and S1074 in the BH is light magenta. Residues in the BH that are close to substitutions in the F loop are magenta. The borders of the F-loop deletion are shown by a black line. (D) Sequence alignments of the TL (green), F loop (red), and BH (magenta) in different RNAPs. Tth indicates T. thermophilus; Eco, E. coli; and Sce, S. cerevisiae RNAPII. The color code is the same as in A–C. Amino acid substitutions in Taq/Dra RNAPs are in red. Residues in the TL that interact with the F loop, and vice versa, are shown with asterisks above the alignments. Residues that interact with Stl in T. thermophilus RNAP (15) are shown on a gray background. The α-helical and loop segments in the closed and open states of the TL are shown by thick and thin lines, respectively, below the TL alignment. Positions of the 188-residue insertion in the TL in E. coli RNAP and the 20-residue insertion in the F loop in RNAPII are indicated by arrowheads below the alignments.

Fig. 2.

Elongation rates of Taq, Dra, G-loop-Dra, and F-Dra RNAPs. The elongation rates were measured as described in Materials and Methods at 40 °C (A) and 20 °C (B). Positions of the halted 26-nt RNA and 157-nt terminated RNA product are shown on the left.

Fig. 3.

The rates of single-nucleotide addition measured for different RNAPs. (A) The structure of the minimal nucleic acid scaffold. (B) Electrophoretic separation of reaction products from a quench-flow assay of wild-type Taq and Dra RNAPs. The reaction was performed on the minimal scaffold containing 5′-labeled 8-nt RNA at 20 °C at 1 mM UTP. (C) Kinetics of UTP incorporation by different RNAPs at 20 °C. The percent of synthesis of the 9-nt RNA product is shown on the ordinate. The reaction was performed with RNAPs from Taq, Dra, mosaic F-Dra, and G-loop-Dra RNAPs, as well as the ΔF-loop Taq RNAP either in the absence or in the presence of Stl (100 μg/mL).

Fig. 4.

Kinetics of RNA cleavage by RNAP from Taq and its mutant variants. (A) The scaffold used in the experiments. The position of RNA cleavage is shown by an arrowhead. (B) Kinetics of RNA cleavage at 20 °C. Positions of the starting 13-nt RNA and 11-nt cleavage product are shown by arrows. (C) Kinetics of RNA cleavage in the presence of Stl (100 μg/mL).

Fig. 5.

Possible conformational changes in the F loop during catalysis. A close-up view of the TL–F-loop interface in the NTP-bound EC (3). The RNAP orientation and the color scheme are the same as in Fig. 1A. Residues Q1033, Q1037, R1042, L1044, Q1046, and E1051 are shown as sticks; residue S1074 is shown as a light magenta sphere. The F loop in the NTP-free EC (1) is superimposed onto the figure; the backbone is shown in light red, and the amino acid side chains are white. Blue spheres indicate positions of the P750L and F773V substitutions in E. coli RNAP (correspond to P1048 and F1071 in Taq), leading to CBR dependence. Red sphere indicates position of the R774C substitution in E. coli RNAP (R1042 in Taq). Gray spheres indicate positions of insertions in the TL in E. coli RNAP (Ins1252) and the F loop in various bacterial lineages and in archaeal and eukaryotic RNAPs (Ins1050).