COMPUTATIONAL STUDIES OF RNA RECOGNITION AND CATALYSIS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 其他生物分子（如蛋白质、辅因子和其他RNA分子）对RNA的准确识别对许多细胞功能至关重要。采用各种计算化学工具，如分子动力学模拟，量子计算和混合量子力学/分子力学方法，我们的研究探讨了三个主要领域，信使RNA-转移RNA（mRNA-tRNA）识别，蛋白质翻译的关键步骤，人类免疫缺陷病毒（HIV）中的蛋白质-RNA相互作用和催化RNA分子的pKa计算。在第一个领域，自然发生的，转录后修饰的碱基在影响tRNA-mRNA识别的作用进行检查。在人tRNALys，3中，我们发现在反密码子碱基中维持典型的阶梯状构象需要37位的修饰碱基（34 - 36）。采用M05 - 2X功能的自然键轨道分析的从头算研究正在进行中，以确定潜在的稳定力和在所有tRNA中保持阶梯状构象的第37位修饰碱基的作用。在M05 - 2X/6 - 31 + G（d，p）理论水平上的氢位置优化需要对四核苷酸和三核苷酸进行（二聚体是~1400个基函数），在我们的本地机器上可能需要超过45天/计算。需要更快的计算资源才能使该项目取得进展。在第二个研究领域，我们正在研究水和静电在RNA肽识别中的作用。在晚期阶段，Rev-RRE识别介导部分和未剪接的HIV mRNA的核质输出。根据Frankel及其同事进行的体外选择研究，已发现称为RSG-1.2的合成肽以比天然Rev肽更大的亲和力和特异性结合RRE。我们模拟了Rev和RSG-1.2肽与RRE RNA在明确的水使用琥珀色，并发现了水的结构之间的相关性肽-RNA复合物和结合亲和力。需要更多的模拟来证实早期的发现。系统大约有35，000个原子，使用并行AMBER代码可以更有效地收集数据。最后，我们与达林约克合作，计算了被称为核酶的催化RNA分子的pKas。热力学积分方法需要平衡的启动系统。目前的系统是在显式溶剂（TIP4Pew）中进行的，包括超过中和的RNA的150mM NaCl缓冲溶液，并且是约75，000个原子。这些系统需要多轮模拟退火来平衡离子气氛，然后在进行TI计算之前，必须在缓冲液存在下随后平衡RNA。这种分配要求利用并行计算设施，同时也为未来的分配要求探索最佳的Teragrid平台。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Accurate RNA recognition by other biomolecules such as proteins, cofactors and other RNA molecules are critical to many cellular functions. Employing a variety of computational chemistry tools such as molecular dynamics simulations, quantum calculations and hybrid quantum mechanical/molecular mechanics methods, our research examines three primary areas, messenger RNA - transfer RNA (mRNA-tRNA) recognition, a key step in the translation of proteins, protein-RNA interactions in human immunodeficiency virus (HIV) and pKa calculations in catalytic RNA molecules. In the first area, the role of naturally occurring, posttranscriptionally modified bases in affecting tRNA-mRNA recognition is examined. In human tRNALys,3, we have found that a modified base at position 37 are required for maintenance of a canonical stair-stepped conformation in the anticodon bases (34-36). Ab initio studies employing natural bond orbital analysis with the M05-2X functional are underway to determine the underlying stabilizing forces and the role of modified bases at the 37th position in retaining a stair-stepped conformation in all tRNAs. Optimization of hydrogen positions at the M05-2X/6-31+G(d,p) theory level needs to be carried out for tetranucleotides and trinucleotides (dimers are ~1400 basis functions), which on our local machines can take greater than 45 days/calculation. Faster computing resources are required to make progress on this project. In the second area of research, we are examining the role of water and electrostatics in RNA-peptide recognition. In late phase Rev-RRE recognition mediates nucleocytoplasmic export of partially and unspliced HIV mRNA. From in vitro selection studies performed by Frankel and coworkers, a synthetic peptide known as RSG-1.2 has been found to bind RRE with greater affinity and specificity than the native Rev peptide. We have simulated both Rev and RSG-1.2 peptides complexed with the RRE RNA in explicit water using AMBER and have found a correlation between water structure in the peptide-RNA complexes and binding affinity. More simulations to corroborate earlier findings are required. Systems are roughly 35,000 atoms and data could be collected more efficiently employing parallel AMBER code. Lastly, in collaboration with Darrin York, we are calculating pKas in catalytic RNA molecules known as ribozymes. The thermodynamic integration methods require equilibrated starting systems. Current systems are carried out in explicit solvent (TIP4Pew), include 150 mM NaCl buffer solution beyond the neutralized RNA and are about 75,000 atoms. These systems require a number of simulated annealing rounds to equilibrate the ion atmosphere and then the RNA must be subsequently equilibrated in the presence of the buffer before TI calculations can be performed. This allocation is requested to take advantage of parallel computing facilities while also exploring optimum Teragrid platforms for future allocation requests.