Réblová Kamila, Střelcová Zora, Kulhánek Petr, Beššeová Ivana, Mathews David H, Nostrand Keith Van, Yildirim Ilyas, Turner Douglas H, Sponer Jiří
Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 61265 Brno, Czech Republic.
J Chem Theory Comput. 2010 Jan 1;2010(6):910-929.
Functional RNA molecules such as ribosomal RNAs frequently contain highly conserved internal loops with a 5'-UAA/5'-GAN (UAA/GAN) consensus sequence. The UAA/GAN internal loops adopt distinctive structure inconsistent with secondary structure predictions. The structure has a narrow major groove and forms a trans Hoogsteen/Sugar edge (tHS) A/G base pair followed by an unpaired stacked adenine, a trans Watson-Crick/Hoogsteen (tWH) U/A base pair and finally by a bulged nucleotide (N). The structure is further stabilized by a three-adenine stack and base-phosphate interaction. In the ribosome, the UAA/GAN internal loops are involved in extensive tertiary contacts, mainly as donors of A-minor interactions. Further, this sequence can adopt an alternative 2D/3D pattern stabilized by a four-adenine stack involved in a smaller number of tertiary interactions. The solution structure of an isolated UAA/GAA internal loop shows substantially rearranged base pairing with three consecutive non-Watson-Crick base pairs. Its A/U base pair adopts an incomplete cis Watson-Crick/Sugar edge (cWS) A/U conformation instead of the expected Watson-Crick arrangement. We performed 3.1 µs of explicit solvent molecular dynamics (MD) simulations of the X-ray and NMR UAA/GAN structures, supplemented by MM-PBSA free energy calculations, locally enhanced sampling (LES) runs, targeted MD (TMD) and nudged elastic band (NEB) analysis. We compared parm99 and parmbsc0 force fields and net-neutralizing Na(+) vs. excess salt KCl ion environments. Both force fields provide a similar description of the simulated structures, with the parmbsc0 leading to modest narrowing of the major groove. The excess salt simulations also cause a similar effect. While the NMR structure is entirely stable in simulations, the simulated X-ray structure shows considerable widening of the major groove, loss of base-phosphate interaction and other instabilities. The alternative X-ray geometry even undergoes conformational transition towards the solution 2D structure. Free energy calculations confirm that the X-ray arrangement is less stable than the solution structure. LES, TMD and NEB provide a rather consistent pathway for interconversion between the X-ray and NMR structures. In simulations, the incomplete cWS A/U base pair of the NMR structure is water mediated and alternates with the canonical A-U base pair, which is not indicated by the NMR data. Completion of full cWS A/U base pair is prevented by the overall internal loop arrangement. In summary, the simulations confirm that the UAA/GAN internal loop is a molecular switch RNA module that adopts its functional geometry upon specific tertiary contexts.
诸如核糖体RNA之类的功能性RNA分子通常含有高度保守的内部环，其具有5'-UAA/5'-GAN（UAA/GAN）共有序列。UAA/GAN内部环呈现出与二级结构预测不一致的独特结构。该结构具有狭窄的大沟，并形成一个反式Hoogsteen/糖边缘（tHS）A/G碱基对，随后是一个未配对的堆积腺嘌呤、一个反式沃森-克里克/霍斯特恩（tWH）U/A碱基对，最后是一个凸起的核苷酸（N）。该结构通过三个腺嘌呤堆积和碱基-磷酸相互作用进一步稳定。在核糖体中，UAA/GAN内部环参与广泛的三级相互作用，主要作为A- minor相互作用的供体。此外，该序列可以采用由四个腺嘌呤堆积稳定的另一种二维/三维模式，参与较少数量的三级相互作用。一个分离的UAA/GAA内部环的溶液结构显示出碱基配对有显著重排，有三个连续的非沃森-克里克碱基对。其A/U碱基对采用不完全的顺式沃森-克里克/糖边缘（cWS）A/U构象，而不是预期的沃森-克里克排列。我们对X射线和核磁共振UAA/GAN结构进行了3.1微秒的显式溶剂分子动力学（MD）模拟，并辅以MM-PBSA自由能计算、局部增强采样（LES）运行、靶向MD（TMD）和推挤弹性带（NEB）分析。我们比较了parm99和parmbsc0力场以及净中和的Na(+)与过量盐KCl离子环境。两个力场对模拟结构提供了相似的描述，parmbsc0导致大沟适度变窄。过量盐模拟也产生类似的效果。虽然核磁共振结构在模拟中完全稳定，但模拟的X射线结构显示大沟显著变宽、碱基-磷酸相互作用丧失以及其他不稳定性。替代的X射线几何结构甚至向溶液二维结构发生构象转变。自由能计算证实X射线排列比溶液结构更不稳定。LES、TMD和NEB为X射线和核磁共振结构之间的相互转换提供了相当一致的途径。在模拟中，核磁共振结构中不完全的cWS A/U碱基对由水介导，并与经典的A-U碱基对交替，这在核磁共振数据中未显示。完全的cWS A/U碱基对的形成受到整个内部环排列的阻碍。总之，模拟证实UAA/GAN内部环是一个分子开关RNA模块，在特定的三级环境下采用其功能几何结构。
