Carr Carolyn E, Marky Luis A
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, Nebraska.
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, Nebraska.
Biophys J. 2017 Aug 8;113(3):529-539. doi: 10.1016/j.bpj.2017.06.024.
Intramolecular three-way junctions are commonly found in both DNA and RNA. These structures are functionally relevant in ribozymes, riboswitches, rRNA, and during replication. In this work, we present a thermodynamic description of the unfolding of DNA intramolecular three-way junctions. We used a combination of spectroscopic and calorimetric techniques to investigate the folding/unfolding thermodynamics of two three-way junctions with a closed (Closed-J) or open (Open-J) junction and their appropriate control stem-loop motifs (GAAATT-Hp, CTATC-Hp, and Dumbbell). The overall results show that both junctions are stable over a wide range of salt concentrations. However, Open-J is more stable due to a higher enthalpy contribution from the formation of a higher number of basepair stacks whereas Closed-J has a defined structure and retains the basepair stacking of all three stems. The comparison of the experimental results of Closed-J and Open-J with those of their component stem-loop motifs allowed us to be more specific about their cooperative unfolding. For instance, Closed-J sacrifices thermal stability of the Dumbbell structure to maintain an overall folded state. At higher salt concentration, the simultaneous unfolding of the above domains is lost, resulting in the unfolding of the three separate stems. In contrast, the junction of Open-J in low salt retains the thermal and enthalpic stability of the Dumbbell structure although sacrificing stability of the CTATC stem. The relative stability of Dumbbell is the primary reason for the higher ΔG°, or free energy, value seen for Open-J at low salt. Higher salt not only maintains thermal stability of the Dumbbell structure in Open-J but causes the CTATC stem to fully fold.
分子内三向连接在DNA和RNA中都很常见。这些结构在核酶、核糖开关、rRNA以及复制过程中具有重要的功能。在这项工作中，我们给出了DNA分子内三向连接解折叠的热力学描述。我们结合使用光谱和量热技术，研究了具有闭合（Closed-J）或开放（Open-J）连接的两个三向连接及其相应的对照茎环基序（GAAATT-Hp、CTATC-Hp和哑铃结构）的折叠/解折叠热力学。总体结果表明，这两种连接在很宽的盐浓度范围内都是稳定的。然而，Open-J更稳定，这是因为形成了更多碱基对堆积，从而有更高的焓贡献，而Closed-J具有明确的结构，并保留了所有三个茎的碱基对堆积。将Closed-J和Open-J的实验结果与其组成茎环基序的实验结果进行比较，使我们能够更具体地了解它们的协同解折叠。例如，Closed-J牺牲了哑铃结构的热稳定性以维持整体折叠状态。在较高盐浓度下，上述结构域同时解折叠的情况消失，导致三个独立茎的解折叠。相比之下，低盐浓度下Open-J的连接保留了哑铃结构的热稳定性和焓稳定性，尽管牺牲了CTATC茎的稳定性。哑铃结构的相对稳定性是低盐浓度下Open-J的ΔG°（即自由能）值较高的主要原因。较高的盐浓度不仅维持了Open-J中哑铃结构的热稳定性，还使CTATC茎完全折叠。
