Roca Jorjethe, Chen Yi-Lan, Woodson Sarah A
T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218.
Proc Natl Acad Sci U S A. 2025 Jul 15;122(28):e2503747122. doi: 10.1073/pnas.2503747122. Epub 2025 Jul 10.
Small noncoding RNAs (sRNAs) regulate gene expression in bacteria in response to diverse stimuli in the environment and the host. Most sRNAs regulate mRNA expression by directly base pairing with complementary sites in the target mRNA with the help of the chaperone protein Hfq. sRNAs and Hfq must rapidly search hundreds of candidate mRNAs for matched (cognate) targets while discriminating against noncognate targets. Here, we use single-molecule fluorescence microscopy to directly observe how cognate and noncognate mRNAs bind immobilized sRNA-Hfq. The results show that initially unstable sRNA-Hfq-mRNA complexes either dissociate within seconds by ejecting one of the two RNAs, depending on their interactions with Hfq, or are stabilized by sRNA-mRNA base pairing. Cognate mRNAs are more likely to form long-lived sRNA-Hfq-mRNA complexes, even in the presence of competing RNA. Active competition for the mutual Hfq chaperone introduces a kinetic barrier to RNA colocalization that is resolved by base pairing, driving the accumulation of cognate sRNA-mRNA interactions while eliminating noncognate interactions.
小非编码RNA（sRNA）可响应环境和宿主中的多种刺激来调节细菌中的基因表达。大多数sRNA借助伴侣蛋白Hfq与靶mRNA中的互补位点直接碱基配对来调节mRNA表达。sRNA和Hfq必须在数百种候选mRNA中快速搜索匹配的（同源）靶标，同时区分非同源靶标。在这里，我们使用单分子荧光显微镜直接观察同源和非同源mRNA如何结合固定化的sRNA-Hfq。结果表明，最初不稳定的sRNA-Hfq-mRNA复合物要么在几秒钟内通过排出两个RNA之一而解离，这取决于它们与Hfq的相互作用，要么通过sRNA-mRNA碱基配对而稳定下来。即使存在竞争性RNA，同源mRNA也更有可能形成长寿命的sRNA-Hfq-mRNA复合物。对共同的Hfq伴侣的主动竞争为RNA共定位引入了动力学障碍，该障碍通过碱基配对得以解决，从而推动同源sRNA-mRNA相互作用的积累，同时消除非同源相互作用。
