Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 1020 Locust St, Philadelphia, PA, 19107, USA.
Nat Commun. 2024 Aug 20;15(1):7134. doi: 10.1038/s41467-024-50817-9.
The mechanism by which RNAP selects cognate substrates and discriminates between deoxy and ribonucleotides is of fundamental importance to the fidelity of transcription. Here, we present cryo-EM structures of human mitochondrial transcription elongation complexes that reveal substrate ATP bound in Entry and Insertion Sites. In the Entry Site, the substrate binds along the O helix of the fingers domain of mtRNAP but does not interact with the templating DNA base. Interactions between RNAP and the triphosphate moiety of the NTP in the Entry Site ensure discrimination against nucleosides and their diphosphate and monophosphate derivatives but not against non-cognate rNTPs and dNTPs. Closing of the fingers domain over the catalytic site results in delivery of both the templating DNA base and the substrate into the Insertion Site and recruitment of the catalytic magnesium ions. The cryo-EM data also reveal a conformation adopted by mtRNAP to reject a non-cognate substrate from its active site. Our findings establish a structural basis for substrate binding and suggest a unified mechanism of NTP selection for single-subunit RNAPs.
RNAP 选择同源底物并区分脱氧核苷酸和核糖核苷酸的机制对转录的保真度至关重要。在这里，我们呈现了人类线粒体转录延伸复合物的冷冻电镜结构，揭示了底物 ATP 结合在入口和插入位点。在入口位点，底物沿着 mtRNAP 的手指结构域的 O 螺旋结合，但不与模板 DNA 碱基相互作用。RNAP 与 NTP 的三磷酸部分之间的相互作用确保了对核苷及其二磷酸和单磷酸衍生物的区分，但不能区分非同源 rNTP 和 dNTP。手指结构域在催化位点上的闭合导致模板 DNA 碱基和底物都被递送到插入位点，并募集催化镁离子。冷冻电镜数据还揭示了 mtRNAP 采用的一种构象，用于从其活性位点排斥非同源底物。我们的发现为底物结合建立了结构基础，并为单亚基 RNAP 对 NTP 的选择提出了一个统一的机制。
