Department of Chemistry, University of Florida, Gainesville, FL, USA.
Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH, USA.
Nat Commun. 2022 Aug 31;13(1):5120. doi: 10.1038/s41467-022-32843-7.
Binding of precursor tRNAs (ptRNAs) by bacterial ribonuclease P (RNase P) involves an encounter complex (ES) that isomerizes to a catalytic conformation (ES*). However, the structures of intermediates and the conformational changes that occur during binding are poorly understood. Here, we show that pairing between the 5' leader and 3'RCCA extending the acceptor stem of ptRNA inhibits ES* formation. Cryo-electron microscopy single particle analysis reveals a dynamic enzyme that becomes ordered upon formation of ES* in which extended acceptor stem pairing is unwound. Comparisons of structures with alternative ptRNAs reveals that once unwinding is completed RNase P primarily uses stacking interactions and shape complementarity to accommodate alternative sequences at its cleavage site. Our study reveals active site interactions and conformational changes that drive molecular recognition by RNase P and lays the foundation for understanding how binding interactions are linked to helix unwinding and catalysis.
前体 tRNA（ptRNA）与细菌核糖核酸酶 P（RNase P）的结合涉及到一个遭遇复合物（ES），该复合物发生异构化形成催化构象（ES*）。然而，结合过程中中间产物的结构和构象变化仍知之甚少。在这里，我们表明，ptRNA 的 5' 前导序列与 3'RCCA 的配对延伸了受体茎，从而抑制了 ES的形成。低温电子显微镜单颗粒分析显示，酶是动态的，在形成 ES时变得有序，其中延伸的受体茎配对被解开。与替代 ptRNA 的结构比较表明，一旦解开完成，RNase P 主要利用堆积相互作用和形状互补来适应切割位点的替代序列。我们的研究揭示了驱动 RNase P 分子识别的活性位点相互作用和构象变化，并为理解结合相互作用如何与螺旋解开和催化相关奠定了基础。
