Biophysics Program, Stanford University, Stanford, CA 94305.
Department of Applied Physics, Stanford University, Stanford, CA 94305;
Proc Natl Acad Sci U S A. 2018 Nov 20;115(47):11976-11981. doi: 10.1073/pnas.1812122115. Epub 2018 Nov 5.
Riboswitches modulate gene expression in response to small-molecule ligands. Switching is generally thought to occur via the stabilization of a specific RNA structure conferred by binding the cognate ligand. However, it is unclear whether any such stabilization occurs for riboswitches whose ligands also play functional roles, such as the ribozyme riboswitch, which undergoes self-cleavage using its regulatory ligand, glucosamine 6-phosphate, as a catalytic cofactor. To address this question, it is necessary to determine both the conformational ensemble and its ligand dependence. We used optical tweezers to measure folding dynamics and cleavage rates for the core ribozyme over a range of forces and ligand conditions. We found that the folding of a specific structural element, the P2.2 duplex, controls active-site formation and catalysis. However, the folded state is only weakly stable, regardless of cofactor concentration, supplying a clear exception to the ligand-based stabilization model of riboswitch function.
Riboswitches 通过响应小分子配体来调节基因表达。通常认为，配体的结合会稳定特定的 RNA 结构，从而实现切换。然而，对于那些配体也具有功能作用的 riboswitches，例如核酶 riboswitch，其使用调节配体葡萄糖胺 6-磷酸作为催化辅因子进行自我切割，其是否发生这种稳定化尚不清楚。为了解决这个问题，有必要确定构象整体及其配体依赖性。我们使用光学镊子在一系列力和配体条件下测量核心核酶的折叠动力学和切割速率。我们发现，特定结构元素 P2.2 双链的折叠控制着活性位点的形成和催化。然而，无论辅因子浓度如何，折叠状态都非常不稳定，这为 riboswitch 功能的基于配体的稳定模型提供了一个明显的例外。
