TY - JOUR
T1 - Effects of individual base-pairs on in vivo target search and destruction kinetics of bacterial small RNA
AU - Poddar, Anustup
AU - Azam, Muhammad S.
AU - Kayikcioglu, Tunc
AU - Bobrovskyy, Maksym
AU - Zhang, Jichuan
AU - Ma, Xiangqian
AU - Labhsetwar, Piyush
AU - Fei, Jingyi
AU - Singh, Digvijay
AU - Luthey-Schulten, Zaida
AU - Vanderpool, Carin K.
AU - Ha, Taekjip
N1 - Funding Information:
We would like to thank Erel Levine, Divya Balasubramanian, and D. Jin for plasmids and strains. We thank Hao Zhang at the Cell Sorting Core Facility (Bloomberg School of Public Health) for helping us with the flow cytometry and sorting experiments. We appreciate and thank Prof. Sarah Woodson for going through the manuscript and providing insightful suggestions. This work was supported by grants from National Institutes of Health R01 GM112659 (M.B., M.S.A., T.H., J.Z., and A.P.), R35 GM122569 (T.H., J.Z., and A.P.), National Science Foundation PHY 1430124 (T.H., Z.L.-S., J.Z., and A.P). T.H. is an investigator with the Howard Hughes Medical Institute.
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/12
Y1 - 2021/12
N2 - 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.
AB - 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.
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U2 - 10.1038/s41467-021-21144-0
DO - 10.1038/s41467-021-21144-0
M3 - Article
C2 - 33558533
AN - SCOPUS:85100991948
VL - 12
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
IS - 1
M1 - 874
ER -