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.T ranscription termination is an essential process in all organisms that releases the RNA chain and DNA template from transcribing RNA polymerase (RNAP). Termination prevents unwanted gene expression (1), recycles RNAP for new initiation events (2), and prevents genome-destabilizing collisions with the replication machinery (3). In bacteria, intrinsic termination of the elongating transcription complex (EC) can occur without accessory factors in response to a signal in the DNA and newly synthesized RNA. Intrinsic termination occurs over a 2-to 3-nt window of EC destabilization (4) and is caused by the formation of a GC-rich RNA hairpin in the RNA exit channel of RNAP, immediately upstream of a 3′-terminal, ∼8-nt U-rich RNA tract (U-tract; Fig. 1A). Precise termination of the highly stable EC requires rapid entry into a termination pathway before continued nucleotide addition moves the EC beyond the region of destabilization (4-6).A thermodynamic termination model posits that the relative free energy barriers to termination versus elongation determine the fraction of ECs that terminate; thus, termination efficiency (TE) is kinetically determined by competing rates of nucleotide addition and termination (5, 7) (Fig. 1B). The EC is stabilized by polar and van der Waals contacts between RNAP and (i) the 9-to ...

RNA polymerase clamp movement aids dissociation from DNA but is not required for RNA release at intrinsic terminators

“…Second, we found that clamp movements are more important for dissociation of RNAP from DNA than for dissociation of RNA from the EC, which is the defining step in termination. Finally, we found that restricting movements of the TL markedly slowed the rates of both RNA and RNAP release, consistent with a role for TL-mediated conformational fluctuations in the termination process [14].…”

“…These effects on elongation, all of which caused corresponding changes in TE, are likely caused primarily by changes in pausing, which were evident in transcription elongation patterns ( Figure S1). Although we cannot exclude effects on on-pathway elongation, it is notable that the D-LF and C-LF disulfides that affect intrinsic termination only indirectly have large and opposite effects on a canonical hairpinstabilized pause [31], and that pausing is known to occur at intrinsic terminators [10,14].…”

“…The lack of inhibition of RNA release by lid-flap crosslinking also provides further reason to doubt the hairpin-TL contact model on intrinsic termination [19]. This model, which also has been questioned on other grounds [14,17,50], requires the terminator hairpin disrupt RNAP interactions by sweeping through the cleft, starting by breaking the lid-flap interaction.…”

“…Intrinsic terminators facilitate entry into the termination pathway through the U-tract and its surrounding sequences, which promote elemental pausing of the EC [10][11][12][13] (Figure 1A). The U-tract pause slows elongation and provides time for the GC-dyad to begin folding into a terminator hairpin (Thp), possibly inducing a transient hairpin-stabilized pause [14]. Unlike conventional hairpin pause signals, the juxtaposition of the weak rU:dA hybrid [15] and additional strong rG:rC base pairs at a terminator allow extension of the hairpin by an additional 2-3 bp, coming to within 8 nt of the RNA 3′ end.…”

“…A second hypothesis is that the Thp, once formed, can invade the main channel to contact the folded trigger loop (TL), disrupting RNAP-NA contacts and forcing clamp opening [19]. Other findings suggest that Thp-TL contact may reflect post-termination RNAP-RNA binding [17] and that termination may proceed through multiple, alternative RNAP conformational states whose formation is aided by TL flexibility [14]. However, the role of clamp motions during steps in the termination mechanism remains unexplored.…”