Program in Cellular, Molecular and Developmental Biology and Biophysics, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
Nucleic Acids Res. 2011 Jul;39(12):5193-202. doi: 10.1093/nar/gkr062. Epub 2011 Mar 4.
The Sm protein Hfq binds small non-coding RNA (sRNAs) in bacteria and facilitates their base pairing with mRNA targets. Molecular beacons and a 16 nt RNA derived from the Hfq binding site in DsrA sRNA were used to investigate how Hfq accelerates base pairing between complementary strands of RNA. Stopped-flow fluorescence experiments showed that annealing became faster with Hfq concentration but was impaired by mutations in RNA binding sites on either face of the Hfq ring or by competition with excess RNA substrate. A fast bimolecular Hfq binding step (∼10(8) M(-1)s(-1)) observed with Cy3-Hfq was followed by a slow transition (0.5 s(-1)) to a stable Hfq-RNA complex that exchanges RNA ligands more slowly. Release of Hfq upon addition of complementary RNA was faster than duplex formation, suggesting that the nucleic acid strands dissociate from Hfq before base pairing is complete. A working model is presented in which rapid co-binding and release of two RNA strands from the Hfq ternary complex accelerates helix initiation 10 000 times above the Hfq-independent rate. Thus, Hfq acts to overcome barriers to helix initiation, but the net reaction flux depends on how tightly Hfq binds the reactants and products and the potential for unproductive binding interactions.
Sm 蛋白 Hfq 在细菌中结合小的非编码 RNA（sRNAs），并促进它们与 mRNA 靶标进行碱基配对。分子信标和源自 DsrA sRNA 中 Hfq 结合位点的 16 个核苷酸 RNA 被用于研究 Hfq 如何加速互补 RNA 链之间的碱基配对。停流荧光实验表明，随着 Hfq 浓度的增加，退火速度加快，但 Hfq 环两面的 RNA 结合位点突变或与过量 RNA 底物竞争会损害退火。用 Cy3-Hfq 观察到快速的双分子 Hfq 结合步骤（∼10(8) M(-1)s(-1)），随后是缓慢的转变（0.5 s(-1)）到稳定的 Hfq-RNA 复合物，其更缓慢地交换 RNA 配体。添加互补 RNA 后 Hfq 的释放速度快于双链体形成速度，这表明在碱基配对完全完成之前，核酸链从 Hfq 上解离。提出了一个工作模型，其中两个 RNA 链从 Hfq 三元复合物中的快速共结合和释放，将螺旋起始速度比 Hfq 非依赖性速度提高了 10000 倍。因此，Hfq 作用是克服螺旋起始的障碍，但净反应通量取决于 Hfq 与反应物和产物的结合紧密程度以及无生产性结合相互作用的潜力。
