Trigger-helix folding pathway and SI3 mediate catalysis and hairpin-stabilized pausing by Escherichia coli RNA polymerase

Abstract

The conformational dynamics of the polymorphous trigger loop (TL) in RNA polymerase (RNAP) underlie multiple steps in the nucleotide addition cycle and diverse regulatory mechanisms. These mechanisms include nascent RNA hairpin-stabilized pausing, which inhibits TL folding into the trigger helices (TH) required for rapid nucleotide addition. The nascent RNA pause hairpin forms in the RNA exit channel and promotes opening of the RNAP clamp domain, which in turn stabilizes a partially folded, paused TL conformation that disfavors TH formation. We report that inhibiting TH unfolding with a disulfide crosslink slowed multiround nucleotide addition only modestly but eliminated hairpin-stabilized pausing. Conversely, a substitution that disrupts the TH folding pathway and uncouples establishment of key TH–NTP contacts from complete TH formation and clamp movement allowed rapid catalysis and eliminated hairpin-stabilized pausing. We also report that the active-site distal arm of the TH aids TL folding, but that a 188-aa insertion in the Escherichia coli TL (sequence insertion 3; SI3) disfavors TH formation and stimulates pausing. The effect of SI3 depends on the jaw domain, but not on downstream duplex DNA. Our results support the view that both SI3 and the pause hairpin modulate TL folding in a constrained pathway of intermediate states.

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

“…The presence of the TL thus favors entry into the elemental paused state in WT ECs, which would extend the kinetic window for T hp formation. The binding of asRNA also had minimal effect on nucleotide addition rate by ΔTL RNAP (Table 2), consistent with previous reports that the TL participates in the hairpin-stabilized paused state (22,37,45). We conclude that, in addition to stimulating termination, the TL helps establish and inhibits escape from paused states at terminators, thereby increasing flux of ECs into the termination pathway.…”

“…However, SI3 had less effect on the rate of A20 addition after asRNA binding (Table 2). Although an approximately threefold effect of SI3 on pausing stabilized by a hairpin extending to −12 has been observed previously (37,49), the closer approach of the duplex stem to the RNA 3′ end may obviate this effect of SI3 at terminators (50). As a consequence of less pausing overall, the TE for ΔSI3 ECs was ∼34% vs. ∼42% for WT ( Table 1), indicating that SI3-mediated pause stimulation increased TE by increasing flux of ECs into the termination pathway, even though the termination rate per se was minimally affected.…”

“…1 D and E), present in many Gram-negative bacterial lineages (46,47). SI3 affects TL function in elongation, pausing, and intrinsic cleavage (37,48,49). A monoclonal antibody (mAb) that binds SI3 was shown to decrease dissociation of ECs stalled at a termination site (28).…”

“…To address these questions, we sought to test termination at t his2 by previously characterized TL variants that either stabilize the folded TL conformation (Ala 6 ) or prevent folding and favor unfolded conformations (LTPP; Fig. 1E) (36,37). For the TH-destabilizing LTPP substitution, we could make a direct comparison between the mutant RNAP and WT using the −10 asRNA (SI Appendix, Fig.…”

“…To investigate the role of the TL in termination, we developed an experimental approach that can separate effects on termination rate from effects on elongation rate, which are known to be significant for TL alterations (36)(37)(38). Whereas traditional TE measurements report an aggregate of effects on termination and elongation, isolated measurement of termination rate in our assay allowed us to determine the effects of TL mutations specifically on the termination mechanism.…”