Controlled interplay between trigger loop and Gre factor in the RNA polymerase active centre

Abstract

The highly processive transcription by multi-subunit RNA polymerases (RNAP) can be interrupted by misincorporation or backtracking events that may stall transcription or lead to erroneous transcripts. Backtracked/misincorporated complexes can be resolved via hydrolysis of the transcript. Here, we show that, in response to misincorporation and/or backtracking, the catalytic domain of RNAP active centre, the trigger loop (TL), is substituted by transcription factor Gre. This substitution turns off the intrinsic TL-dependent hydrolytic activity of RNAP active centre, and exchanges it to a far more efficient Gre-dependent mechanism of RNA hydrolysis. Replacement of the TL by Gre factor occurs only in backtracked/misincorporated complexes, and not in correctly elongating complexes. This controlled switching of RNAP activities allows the processivity of elongation to be unaffected by the hydrolytic activity of Gre, while ensuring efficient proofreading of transcription and resolution of backtracked complexes.

Figure 1.

(A) Upon folding the TL forms part of the RNAP active centre. The TL of T. thermophilus RNAP [PDB 2BE5 and 2O5J, respectively (7,34)], which has complete conservation with the active centre amino acids of T. aquaticus RNAP used in our study, in unfolded and folded states is coloured green and cyan, respectively. The amino acids of the TL that form the ‘TL active centre’ upon TL folding (M1238, R1239, H1242, T1243) are shown in space fill in both folded and unfolded states of the TL. The amino acids (not belonging to the TL) that surround catalytic Mg²⁺ ions and the incoming NTP are shown in grey spacefill. The aspartate triad is shown as purple sticks. Mg²⁺ ions of the active centre are shown as spheres. Incoming NTP, RNA 3′-end and DNA template bases are shown as orange, red and black sticks, respectively. The N-terminal coiled-coil domain of Gre factor (structure of E. coli GreA was used; PDB 1GRJ) which, according to our results, substitutes for the TL upon misincorporation or backtracking (see main text), was positioned to reach the active centre of RNAP without significant clashes with surrounding structure, and is shown as yellow ribbons. (B) Elongation complexes used in this study. Shown are the sequences of RNA, template strand (T DNA) and non-template strand (NT DNA) used to assemble elongation complexes (see ‘Materials and Methods’ section). (C) Gre-catalysed hydrolysis is independent of the TL. Kinetics of the cleavage reaction (also schematically shown above the plots) in mEC15 (Figure 1B) by WT RNAP (red) and ΔTL RNAP (blue), in the absence (left) or presence (right) of Gre factor. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). The grey arrows represent the contributions of the TL and Gre to the rate of the reaction. Representative gels for cleavage by WT and ΔTL RNAPs in the absence or the presence of Gre are shown below the plots. Note that the kinetics of Gre catalysed reactions were measured at 20°C to reduce the rate of the reaction to allow it to be measured manually. (D) Gre-catalysed hydrolysis is independent of the H1242 of the TL. Kinetics of the cleavage reaction in mEC15 (as in panel C) by H1242A RNAP with and without Gre, and WT RNAP with Gre. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). (E) The TL is not involved in Mg²⁺ chelation during phosphodiester bond hydrolysis. Dependence of k_obs on the Mg²⁺ concentration during intrinsic cleavage (as in panel C) by WT (red) and ΔTL (blue) RNAPs. The values of apparent K_m[Mg²⁺] are shown below the plots. The values of k_obs at different Mg²⁺ concentrations were calculated by single exponential fitting of the kinetics data (as in panel C) and K_m[Mg²⁺] values were calculated as described in ‘Materials and Methods’ section. (F) pH profiles of the reactions described in panel C in the absence and the presence of Gre. The values of k_obs at different pH were calculated by single exponential fitting of the kinetics data (see panel C and ‘Materials and Methods’ section). Note that reactions with Gre were performed at 20°C (as in panel C).

Figure 2.

Substitution of the TL by Gre depends on the state of the elongation complex. (A) Kinetics of the cleavage reaction (schematically shown above the plots) in mEC15 (Figure 1B) by WT RNAP (left) and ΔTL RNAP (right) in the absence (red) or presence (blue) of the catalytically inactive mutant Gre factor, Gre^D42A/E45A. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section). (B) Kinetics of single nucleotide addition (1 µM and 1 mM CTP; reaction schematically shown above the plots) in cEC15 (Figure 1B) by WT RNAP in the absence (red) or presence (blue) of Gre^D42A/E45A. Solid curves are the single (without Gre^D42A/E45A) or double (with Gre^D42A/E45A) exponential fits of the kinetics data (see ‘Materials and Methods’ section). Blue vertical lines in the left plot show the inhibited (slow) and non-inhibited (fast) fractions of elongation complexes. (C) Kinetics of pyrophosphorolysis and second phosphodiester bond hydrolysis in cEC13 and cEC15. RNAs longer than 13 nt in lanes 2–4 originate from incorporation of NTPs, which are the result of pyrophosphorolysis. (D) Exo III footprinting of the front edge of RNAP in cEC13 and cEC15. Non-template DNA strand (NT DNA) and RNA were labelled with P³² at the 5′-end (asterisks in the scheme on the left). Three different concentrations of Exo III were used for accurate comparison of relative distribution of translocation states in cEC13 and cEC15. The lower panel originates from the same gel as the top panel, and shows the transcripts in the complexes. Black vertical lines in both panels separate lanes originating from the same gel that were brought together. (E) Kinetics of single nucleotide addition (1 µM GTP, reaction schematically shown above the plots) in cEC13 (Figure 1B) by WT RNAP in the absence (red) or presence (blue) of the Gre^D42A/E45A. Solid curves are the single exponential fits of the kinetics data (see ‘Materials and Methods’ section).

Figure 3.

Two models of switching of the active centres, the ‘TL active centre’ (red) and ‘Gre active centre’ (blue), in the RNA polymerase catalytic site (yellow circle). Gre does not interfere with the TL during productive elongation. However, in response to misincorporation or occasional backtracking Gre substitutes for the TL in the active site where it activates the hydrolytic activity to efficiently resolve backtracked complexes. After resolution of backtracked complex is accomplished, Gre is replaced back by the TL allowing continuation of elongation. Switching may take place without (A) or with (B) dissociation of Gre from the elongation complex.