Rapid binding and release of Hfq from ternary complexes during RNA annealing

Abstract

The Sm protein Hfq binds small non-coding RNA (sRNAs) in bacteria and facilitates their base pairing with mRNA targets. Molecular beacons and a 16 nt RNA derived from the Hfq binding site in DsrA sRNA were used to investigate how Hfq accelerates base pairing between complementary strands of RNA. Stopped-flow fluorescence experiments showed that annealing became faster with Hfq concentration but was impaired by mutations in RNA binding sites on either face of the Hfq ring or by competition with excess RNA substrate. A fast bimolecular Hfq binding step (∼108 M−1s−1) observed with Cy3-Hfq was followed by a slow transition (0.5 s−1) to a stable Hfq–RNA complex that exchanges RNA ligands more slowly. Release of Hfq upon addition of complementary RNA was faster than duplex formation, suggesting that the nucleic acid strands dissociate from Hfq before base pairing is complete. A working model is presented in which rapid co-binding and release of two RNA strands from the Hfq ternary complex accelerates helix initiation 10 000 times above the Hfq-independent rate. Thus, Hfq acts to overcome barriers to helix initiation, but the net reaction flux depends on how tightly Hfq binds the reactants and products and the potential for unproductive binding interactions.

C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA

“…Hfq102 had little effect on the amount of duplex formed by Target and Target-U6 (• and ■ in Fig. 1C), consistent with release of the target·beacon duplex from Hfq (39). However, Hfq65 lowered the amount of duplex formed (○ and △ in Fig.…”

“…Although the CTDs made no difference to the rate of RNA binding, we discovered that the CTD is essential for rapid release of the dsRNA product, which normally occurs after the two strands base pair (39) (Fig. 1A).…”

“…We measured base pairing between a fluorescent RNA molecular beacon and a 16-nt Target RNA that is complementary to the loop of the molecular beacon but lacks a specific binding site for Hfq (41). Previous studies showed that the beacon and various target RNAs bind Hfq rapidly (39), forming a ternary complex that accelerates nucleation and zippering of the RNA duplex (18,40) (Fig. 1A).…”

“…By contrast, when a complex of Hfq65-Cy3·D16-FAM was challenged with R16, the fluorescence increased slightly rather than decreased, suggesting that D16-FAM RNA remained bound to Hfq65. Because Hfq65 is an active chaperone and the increase in fluorescence occurs on the same timescale as duplex release from Hfq102-Cy3, it likely depends on annealing of the two RNAs (18,39).…”

“…Target RNAs (Dharmacon) and R16 (Invitrogen) have been previously described (39,41) and were purified by denaturing PAGE. The molecular beacon, D16-FAM, and A18-FAM were obtained from Trilink Biotechnologies and purified by reverse-phase HPLC.…”