JILA, University of Colorado Boulder and National Institute of Standards and Technology, Boulder, Colorado 80309, United States.
Department of Chemistry, University of Colorado Boulder, Boulder, Colorado 80309, United States.
J Phys Chem B. 2022 Jan 13;126(1):69-79. doi: 10.1021/acs.jpcb.1c07833. Epub 2021 Dec 27.
Riboswitches play an important role in RNA-based sensing/gene regulation control for many bacteria. In particular, the accessibility of multiple conformational states at physiological temperatures allows riboswitches to selectively bind a cognate ligand in the aptamer domain, which triggers secondary structural changes in the expression platform, and thereby "switching" between on or off transcriptional or translational states for the downstream RNA. The present work exploits temperature-controlled, single-molecule total internal reflection fluorescence (TIRF) microscopy to study the thermodynamic landscape of such ligand binding/folding processes, specifically for the lysine riboswitch. The results confirm that the riboswitch folds via an induced-fit (IF) mechanism, in which cognate lysine ligand first binds to the riboswitch before structural rearrangement takes place. The transition state to folding is found to be enthalpically favored (Δ < 0), yet with a free-energy barrier that is predominantly entropic (-Δ > 0), which results in folding (unfolding) rate constants strongly dependent (independent) of lysine concentration. Analysis of the single-molecule kinetic "trajectories" reveals this rate constant dependence of on lysine to be predominantly entropic in nature, with the additional lysine conferring preferential advantage to the folding process by the presence of ligands correctly oriented with respect to the riboswitch platform. By way of contrast, van't Hoff analysis reveals enthalpic contributions to the overall folding thermodynamics (Δ) to be surprisingly constant and robustly independent of lysine concentration. The results demonstrate the crucial role of hydrogen bonding between the ligand and riboswitch platform but with only a relatively modest fraction (45%) of the overall enthalpy change needed to access the transition state and initiate transcriptional switching.
Riboswitches 在基于 RNA 的传感/基因调控控制中对许多细菌起着重要作用。特别是，在生理温度下多个构象状态的可及性允许核糖开关选择性地结合适体结构域中的同源配体，这触发了表达平台的二级结构变化，从而在下游 RNA 上“切换”转录或翻译状态的开启或关闭。本工作利用温度控制的单分子全内反射荧光（TIRF）显微镜研究这种配体结合/折叠过程的热力学景观，特别是对于赖氨酸核糖开关。结果证实，核糖开关通过诱导契合（IF）机制折叠，其中同源赖氨酸配体首先与核糖开关结合，然后发生结构重排。发现折叠的过渡态有利于焓（Δ<0），但自由能垒主要是熵（-Δ>0），这导致折叠（解折叠）速率常数强烈依赖（独立）于赖氨酸浓度。对单分子动力学“轨迹”的分析表明，这种对赖氨酸的速率常数依赖性主要是熵性质的，额外的赖氨酸通过配体相对于核糖开关平台正确取向赋予折叠过程优先优势。相比之下，范特霍夫分析表明，整体折叠热力学（Δ）中的焓贡献出人意料地恒定且与赖氨酸浓度独立稳健。结果表明，配体与核糖开关平台之间的氢键起着至关重要的作用，但仅需要总焓变化的相对较小部分（45%）即可达到过渡态并启动转录开关。
