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
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Nat Commun. 2021 Feb 8;12(1):874. 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, and increase the dissociation rate, k, showing that Hfq directly facilitates sRNA-mRNA annealing in vivo. Single base-pair mismatches in the annealing region reduce k by 24-31% and increase k 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.
碱基配对相互作用介导了许多分子间的靶标识别事件。即使单个碱基对错配也会导致活性上的显著差异，但这种变化如何影响体内的靶标搜索动力学尚不清楚。在这里，我们使用高通量测序和定量超分辨率成像来探测细菌小RNA SgrS的突变体及其对ptsG mRNA靶标的调控。破坏伴侣蛋白Hfq结合且位于mRNA退火区域远端的突变仍会降低靶标结合速率k，并增加解离速率k，表明Hfq在体内直接促进sRNA-mRNA退火。退火区域中的单个碱基对错配使k降低24 - 31%，并使k增加14 - 25%，将找到并摧毁靶标的时间延长了约三分之一。破坏连续碱基配对的影响比热力学预期的要小得多，这表明Hfq缓冲了碱基对的破坏。
