Trigger loop dynamics can explain stimulation of intrinsic termination by bacterial RNA polymerase without terminator hairpin contact

Abstract

In bacteria, intrinsic termination signals cause disassembly of the highly stable elongating transcription complex (EC) over windows of two to three nucleotides after kilobases of RNA synthesis. Intrinsic termination is caused by the formation of a nascent RNA hairpin adjacent to a weak RNA-DNA hybrid within RNA polymerase (RNAP). Although the contributions of RNA and DNA sequences to termination are largely understood, the roles of conformational changes in RNAP are less well described. The polymorphous trigger loop (TL), which folds into the trigger helices to promote nucleotide addition, also is proposed to drive termination by folding into the trigger helices and contacting the terminator hairpin after invasion of the hairpin in the RNAP main cleft [Epshtein V, Cardinale CJ, Ruckenstein AE, Borukhov S, Nudler E (2007) Mol Cell 28:991-1001]. To investigate the contribution of the TL to intrinsic termination, we developed a kinetic assay that distinguishes effects of TL alterations on the rate at which ECs terminate from effects of the TL on the nucleotide addition rate that indirectly affect termination efficiency by altering the time window in which termination can occur. We confirmed that the TL stimulates termination rate, but found that stabilizing either the folded or unfolded TL conformation decreased termination rate. We propose that conformational fluctuations of the TL (TL dynamics), not TL-hairpin contact, aid termination by increasing EC conformational diversity and thus access to favorable termination pathways. We also report that the TL and the TL sequence insertion (SI3) increase overall termination efficiency by stimulating pausing, which increases the flux of ECs into the termination pathway.

Keywords: Escherichia coli; RNA polymerase; intrinsic termination; transcription; trigger loop.

Fig. 1.

Intrinsic transcription termination and the polymorphous TL. (A) (Left) The main elements of a canonical intrinsic terminator are shown: a U-rich tract, immediately preceded by a GC-rich T_hp structure. (Right) The T_hp can be mimicked by annealing an asRNA (antisense RNA; dark red) complementary to the nascent RNA transcript (light red) to create a GC-rich duplex. (B) The thermodynamic model for termination (–6), in which the relative free energy barriers to elongation (blue) versus termination (red) determine the probability of each event. The stabilities of the RNA−DNA hybrid (pink) and RNAP−NA interactions (red) are proportional to the arrow height. (C) The steps of intrinsic termination. Steps that lead to termination are indicated by red arrows; steps that lead to terminator bypass are indicated by blue arrows. RNA, red; DNA, black; RNAP, gray. (D) Structure of an EC and relevant elements generated as described in ref. . RNAP is shown as a white surface with modules of interest shown as Cα backbone traces. The alternative structures of the folded TH (orange) and the unfolded TL (green) structures are both shown. For clarity, the TL insertion (SI3, pink) is shown connected to the unfolded TL conformation. (E) The TH and unfolded TL conformations. Blue and red spheres indicate residues mutated in the folded TL stabilizing (Ala₆) and folded TL destabilizing (LTPP) RNAP mutants, respectively.

Fig. 2.

Measuring termination by elongation-compromised RNAP mutants. (A) The t_his2 terminator sequence and asRNAs are shown. The −8 asRNA and −10 asRNA pair at their 5′ ends to the −8 and −10 RNA bases, respectively (where the 3′ nt of U19 RNA is −1). The asterisk denotes the radiolabel at C18 in the nascent RNA. Termination is observed at two sites on t_his2, C18 and U19. (B) Key steps in the t_his2 termination assay. ECs are reconstituted at G17, radiolabeled at C18, and extended past U19 in the presence or absence of asRNA. (C) WT and ΔTL ECs elongate past the termination sites in the absence of asRNA, but terminate efficiently in its presence. WT and ΔTL C18 ECs were assembled as depicted in B. WT ECs were extended with 100 μM UTP and 10 μM ATP for 32 min in the presence or absence of 50 μM −8 asRNA. ΔTL C18 ECs were extended with 10 mM UTP and ATP, and 50 μM −8 asRNA added after 280 s to allow initial U19 accumulation. ΔTL reactions were incubated for 128 min to ensure completion. For WT ECs, TE was calculated as the percent of U19 ECs that terminated, i.e., TE = U19/(U19 + A20⁺) × 100. To account for delayed asRNA addition for ΔTL ECs, TE for ΔTL ECs was calculated as the percent of U19 RNA present at the time of asRNA addition that failed to extend to A20⁺. (D) Robust termination and release of C18 and U19 RNAs occurs with oligos complementary to the exiting RNA (asDNA/asRNA), but not with noncomplementary oligos (nasDNA/nasRNA). G17 ECs were tethered to paramagnetic Co²⁺ beads via a His₁₀ tag on RNAP. The RNAs were radiolabeled at C18 and then extended through U19 with 100 μM UTP and 10 μM ATP in the absence or presence of 50 μM antisense or noncomplementary control oligo. The asRNA is equivalent to −8 asRNA in A. Release efficiency (RE) was calculated as the amount of C18 and U19 RNA that is released in the supernatant (S) as a fraction of total RNA present in the whole reaction (W). W = pellet (RNAP-bound RNA) + S (released RNA).

Fig. 3.

Limited hairpin extension enabled termination rate measurements for slow ΔTL RNAP mutant. (A) Experimental scheme for measurement of the elongation and termination rates of WT and ΔTL ECs. (B) Representative denaturing RNA gel image and (C) reaction progress curves showing the conversion of RNAs from C18 to U19 to A20⁺ for WT ECs in the absence of asRNA. The ΔTL gel image and reaction progress curves are shown in SI Appendix, Fig. S2 A and B. (D and E) Reaction progress curves for (D) WT ECs with −10 asRNA and (E) ΔTL ECs with −10 asRNA; the corresponding gel images are shown in SI Appendix, Fig. S2 C and D, respectively. The time of asRNA addition is indicated with arrows. Error bars represent SD from three or more independent experimental replicates; error bars are smaller than the data markers in some cases.

Fig. 4.

Determination of elongation rates in the absence of asRNA by kinetic fitting. Kinetic models, the corresponding fits, and the rate constants obtained from the fits are shown for (A) WT and (B) ΔTL ECs in the absence of asRNA. Residuals between measured EC amounts and the amounts predicted by the kinetic mechanisms and rate constants from global fits are shown in the lower panels. C18_e, U19_e, and A20⁺ denote elongating ECs with C18, U19, or A20⁺ RNAs, respectively; C18_p and U19_p, elemental paused ECs; U19_t, terminated ECs. Error bars represent SD from three or more independent experimental replicates (Methods); error bars are smaller than the symbols in some cases.

Fig. 5.

The TL increases termination rate. Kinetic models, the corresponding fits, and the rate constants obtained from the fits are shown for (A) WT and (B) ΔTL ECs with −10 asRNA. Time of asRNA addition is indicated with arrows, and is accounted for in the kinetic fits. Residuals between measured EC amounts and the amounts predicted by the kinetic mechanisms and rate constants from global fits are shown in the lower panels. C18_e and U19_e denote elongating ECs with C18 or U19 RNAs, respectively; C18_p and U19_p, elemental paused ECs; asR, free asRNA; C18_asR and U19_asR, asRNA-bound ECs; C18_t and U19_t, terminated ECs; A20⁺, ECs with A20⁺ RNA. Error bars represent SD from three or more independent experimental replicates (Methods); error bars are smaller than the symbols in some cases.

Fig. 6.

TL dynamics increase conformational states of an EC and paths to termination. (A) The MS-MP model of intrinsic termination. Terminating ECs may exist in a family of conformational states with varying stabilities and access to multiple paths to termination. A WT EC with a dynamic TL occupies a diverse distribution of states, enabling access to many paths of EC inactivation (left). The limited EC dynamics of ΔTL ECs (or ECs with restricted TL flexibility) decrease the number of available paths to EC inactivation (right). Thick arrows indicate paths that are more favorable for termination. (B) Hypothetical energy landscape depicting the role of TL conformation in allowing the EC to sample various pathways to termination with differing energy barriers. Blue and red arrows for hairpin pause escape and EC inactivation, respectively, represent the corresponding steps in A. The white dashed arrow indicates a hypothetical low-energy barrier that could be more easily traversed by ECs; the yellow arrow denotes the region of the 3D projection analogous to the yellow arrow in Inset. (Inset) Hypothetical energy diagram depicting the role of TL conformation in altering EC stability.