Analysis, Simulation and Characterization of RNA Motifs

描述(申请人提供)：我们的总体目标是将序列数据与核糖体RNA(5S、16S和23S样RNA)的原子分辨率3D结构相结合，从而生成充分利用序列和3D数据的准确结构比对。最终结果将是开发新的在线软件工具，用于自动化将新序列添加到结构比对序列数据库中的过程，并用于评估新基序的这些比对。由此产生的比对将提高核糖体RNA在各级系统发育研究中的效用，并将为在晶体结构不可用的情况下对具有新兴医学意义的生物的核糖体进行3D建模提供基础。这项建议旨在实现实现总体目标所需的特定子目标，在前一个赠款期间取得的成就的基础上，包括创建一个全面但易于理解的非沃森-克里克基对的分类和汇编(在线提供)，以及一种系统的方法来注释、分类和比对RNA基序，定义为非沃森-克里克基对的有序阵列。子目标包括：1)对核糖体亚基的原子分辨结构中的所有RNA基序进行注释、分类和编目。2)古细菌(H.marismortui)和细菌(D.Radiodurans)5S和23S序列的结构比对(我们有原子分辨3D结构)，以确定保守特征、基序互换和变异点。3)等位度矩阵的比较(I.M.)通过对核糖体RNA中的序列变化进行结构分析，反复提炼I.M.‘S，并确定比对中需要修改的区域。4)识别重复基序的“序列特征”，并对常见的序列变化进行分子动力学模拟，以完善由等位素建模导出的三维结构模型。5)通过重复基序介导的核糖体相互作用的热力学研究。基序将被模块插入到我们设计的用于研究核糖体-RNA自组装的计算机分子中。紫外线熔融实验、等温滴定量热法和圆二色谱将被用来表征相互作用随温度和镁浓度的变化。

DESCRIPTION (provided by applicant): Our overall goal is to integrate sequence data with atomic-resolution 3D structures of the ribosomal RNAs (5S, 16S-like, and 23S-like rRNAs), so as to generate accurate structural alignments that make full use of both the sequence and 3D data. The end result will be the development of new on-line software tools for automating the process of adding new sequences to the database of structurally aligned sequences and for evaluating those alignments for novel motifs. The resulting alignments will improve the utility of the ribosomal RNAs for phylogenetic studies at all levels and will provide a basis for 3D modeling of the ribosomes of organisms of emerging medical interest where crystal structures are not available. This proposal aims to accomplish specific subgoals required for achieving the overall goal, building on the accomplishments of the previous grant period, which include the creation of a comprehensive yet easily understood classification and compilation of non-Watson-Crick basepairs (made available on-line) and a systematic approach to annotating, classifying, and aligning RNA motifs, defined as ordered arrays of non-Watson-Crick basepairs. The subgoals include: 1) Annotating, classifying, and cataloguing all RNA motifs in the atomic-resolution structures of ribosomal subunits. 2) Structural alignment of the archaeal (H. marismortui) and bacterial (D. radiodurans) 5S and 23S sequences (for which we have atomic-resolution 3D structures) to identify conserved features, motif swaps, and points of variation. 3) Comparison of Isostericity Matrices (I.M.) derived from structural analysis with sequence variations in the ribosomal RNAs to iteratively refine the I.M.'s and identify regions in the alignments which require reworking 4) Identification of "sequence signatures" of recurrent motifs and Molecular Dynamics simulation of commonly occurring sequence variations to refine 3D structural models deduced by isosteric modeling. 5) Thermodynamic studies of RNA-RNA interactions mediated by recurrent motifs. Motifs will be modularly inserted in tecto-RNA molecules we have computer designed to study RNA-RNA self-assembly. UV melting experiments, Isothermal Titration Calorimetry and CD spectroscopy will be employed to characterize interactions as functions of temperature and magnesium concentration.