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°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 ...

CBR antimicrobials inhibit RNA polymerase via at least two bridge-helix cap-mediated effects on nucleotide addition

“…The tip of the folded TL is accommodated in a pocket surrounded by conserved structural elements of the RNAP, including the F loop that immediately precedes the BH in the β′ sequence. Alterations to the F loop have profound effects on the RNAP NAC, likely through effects on TL folding (8)(9)(10).…”

“…3A), the F-loop sequences are more diverse (52% identical between E. coli and thermus). Indeed, swapping F-loop sequences between different bacterial RNAPs can confer distinct transcription elongation properties that correspond to the source of the F-loop sequence (8). The F-loop residue E. coli β′I755 forms a part of the CBR binding pocket and makes direct, nonpolar interactions with the CBR compounds (Fig.…”

“…Single amino acid substitutions in the RNAP β and β′ subunits that conferred resistance to CBR compounds (CBR R ) defined a pocket near the RNAP surface bounded by β′ residues of the F loop and the N-terminal part of the bridge helix (BH), and β residues neighboring fork-loop 2 (FL2) and the βDII loop, all RNAP structural elements shown to play important roles in the enzyme's nucleotide addition cycle (NAC) (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). The genetically defined CBR site is ∼31 Å from the RNAP active site Mg 2+ ion and is also distinct from the binding sites of other well-characterized bacterial RNAP inhibitors (SI Appendix, Table S1).…”

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

“…The experiments were performed with the minimal nucleic scaffold template that is bound by RNAP in the posttranslocated state and was previously used extensively to characterize catalysis by Dra RNAP (27)(28)(29) (Fig. S3A).…”

Bacterial Transcription as a Target for Antibacterial Drug Development

“…Residues in the amino-terminal extension of the BH, the F-loop, and the ␤-link (Fig. 1C) contact the TL directly and stabilize its refolding (93).…”