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

• Published in: Nature communications Vol. 12; no. 1; p. 874
• Main Authors: Poddar, Anustup, Azam, Muhammad S., Kayikcioglu, Tunc, Bobrovskyy, Maksym, Zhang, Jichuan, Ma, Xiangqian, Labhsetwar, Piyush, Fei, Jingyi, Singh, Digvijay, Luthey-Schulten, Zaida, Vanderpool, Carin K., Ha, Taekjip
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 08-02-2021; Nature Publishing Group; Nature Portfolio
• Subjects: 13/31; 14/32; 14/63; 631/337/2265; 631/337/384/521; 631/80/2373/2238; Annealing; Bacteria; Base Pairing - genetics; Base Sequence; Escherichia coli - genetics; Gene Dosage; Genes, Reporter; Humanities and Social Sciences; Image resolution; Imaging, Three-Dimensional; In vivo methods and tests; Kinetics; mRNA; multidisciplinary; Mutation; Mutation - genetics; Next-generation sequencing; Nucleotides - genetics; RNA Stability; RNA, Bacterial - genetics; RNA, Messenger - genetics; RNA, Messenger - metabolism; Science; Science (multidisciplinary); Target recognition; Time Factors
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-21144-0

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,
k
on
, and increase the dissociation rate,
k
off
, showing that Hfq directly facilitates sRNA–mRNA annealing in vivo. Single base-pair mismatches in the annealing region reduce
k
on
by 24–31% and increase
k
off
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.
Bacterial small RNA SgrS binds and regulates its primary target,
ptsG
mRNA. Here the authors employ Sort-Seq and super resolution imaging to investigate in vivo target recognition and rejection kinetics of SgrS.