Effects of individual base-pairs on in vivo target search and destruction kinetics of bacterial small RNA

Abstract

Base-pairing interactions mediate many intermolecular target recognition events. Even a single base-pair mismatch can cause a substantial difference in activity but how such changes influence the target search kinetics in vivo is unknown. Here, we use high-throughput sequencing and quantitative super-resolution imaging to probe the mutants of bacterial small RNA, SgrS, and their regulation of ptsG mRNA target. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decrease the rate of target association, kon, and increase the dissociation rate, koff, showing that Hfq directly facilitates sRNA–mRNA annealing in vivo. Single base-pair mismatches in the annealing region reduce kon by 24–31% and increase koff by 14–25%, extending the time it takes to find and destroy the target by about a third. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq buffers base-pair disruptions.

Sequence specificity in DNA binding is mainly governed by association

“…The coupling between association and dissociation rates proposed in this work holds for all bimolecular association-dissociation processes adhering to detailed balance, where a step of rapid testing for molecular recognition precedes the strong binding of a target. Indeed, the anti-correlated relationship between association and dissociation has been observed previously for numerous other systems when perturbing the sequence or salt condition (16,(20)(21)(22). We therefore believe that our theoretical result is very likely to be generally applicable to a wide range of kinetic systems in addition to the ones investigated here, including processes that do not involve protein-DNA interactions.…”

The 3′UTR‐derived sRNA RsaG coordinates redox homeostasis and metabolism adaptation in response to glucose‐6‐phosphate uptake in Staphylococcus aureus

“…RsaG is different from E. coli SgrS, which protects the cell against glucose-phosphate stress and from the depletion of glycolytic intermediates (Richards et al, 2013). Interestingly, a hierarchical regulation mediated by SgrS has been demonstrated (Bobrovskyy et al, 2019;Poddar et al, 2021). At the low level, SgrS represses ptsG-encoded glucose transporter and activates a sugar phosphatase, which promotes dephosphorylation and efflux of phosphosugars.…”

Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities

“…ssl- sRNAs designed with different activities could be easily interfaced synergistically with constitutive and inducible promoters to achieve more tunable regulation performance ( Fig. 5 ) because gene regulation activities are determined not only by sRNAs themselves but also by the concentrations of the target mRNA or Hfq protein [ 13 , 48 , 49 ].…”