Comparative Analysis of Structure in RNAs of Related RNA Coliphages

这里提出的研究来自首席研究员的早期工作，该研究表明，通过电子显微镜可以在从RNA大肠杆菌M12和MS2获得的RNA中看到大量可重复的结构特征。这些研究表明，电子显微镜在RNA中看到的结构可能代表了异常稳定的碱基切割区域。这些结构在病毒遗传图谱中的位置进一步表明，它们可能在调节感染细胞中的病毒基因表达方面发挥作用。最近，PI一直在用电子显微镜检查相关的大肠杆菌RNA的结构，以确定保守的结构特征；保守的特征有望在感染细胞中具有功能意义。对MS2、GA、Qbeta和SP噬菌体基因组RNA的构象进行了研究。这些噬菌体代表4个主要的病毒群。在所有四个RNA中都有一个大的中央环，并在核苷酸水平上建立了一个模型，用于Qbeta中该环的780个核苷酸。该环的结构已通过生化方法得到确认。该环是所有噬菌体RNA在电子显微镜下看到的最稳定的特征之一(抗变性)。这一环的结构和功能将通过遗传分析进行更详细的研究。此外，PI建议使用电子显微镜、比较序列分析、生化探测和计算机模拟在核苷酸水平上为相关的大肠杆菌噬菌体SP、Qbeta和MX1的RNA建立完整的模型。这些遗传学研究是与纽约州立大学下州医学中心微生物学和免疫学部唐·米尔斯合作完成的。米尔斯实验室已经开发出一种遗传系统，通过提供反式野生型病毒蛋白来分离和生长携带大量缺失的Qbeta突变株。研究表明，病毒复制酶基因某些区域的缺失不能与反式提供的病毒复制酶互补，这些区域被称为顺式作用的RNA元件。PI认为，这些突变体定义了基因组中RNA结构重要的区域。突变RNA的构象将通过电子显微镜和生化方法进行检查。此外，这种类型的分析将扩展到上述保守的中央环区。希望通过我们的分析所获得的信息将被证明不仅有助于理解结构在大肠杆菌RNA中的功能意义，而且将有助于理解RNA结构在其他基因组病毒RNA和细胞信使RNA中的作用。

The studies proposed here come from early work by the principal investigator showing that large reproducible structural features could be seen by electron microscopy in RNA obtained from the RNA coliphages M12 and MS2. These studies suggested that the structures that were seen by electron microscopy in the RNA might represent base pared regions that were unusually stable. The location of these structures within the viral genetic map further suggested that they might play a role in regulating viral gene expression in infected cells. Recently the PI has been examining the structure of related coliphage RNAs by electron microscopy in order to identify conserved structural features; conserved features are expected to have functional significance in infected cells. The conformation of genomic RNAs from the bacteriophages MS2, GA, Qbeta and SP were examined. These bacteriophages represent 4 major viral groups. A large central loop is conserved in all four RNAs and a model was developed at the nucleotide level for 780 nucleotides of this loop in Qbeta. The structure of the loop has been confirmed using biochemical methods. The loop is one of the most stable features (resistant to denaturation) seen by electron microscopy in all of the phage RNAs. The structure and function of this loop will be studied in more detail by genetic analysis. In addition, the PI proposes to develop full models at the nucleotide level for the RNAs of the related coliphages SP, Qbeta and MX1 using electron microscopy, comparative sequence analysis, biochemical probing, and computer modeling. The genetic studies are being done in collaboration with Don Mills, Department of Microbiology and Immunology, SUNY Downstate Medical Center. The Mills lab has developed a genetic system for isolating and growing mutants of Qbeta carrying large deletions by supplying the wild type viral protein in trans. The have shown that deletions in some regions of the viral replicase gene cannot be complemented with viral replicase that is supplied in trans and have called these regions cis-acting RNA elements. The PI believes that these mutants define regions in the genome where the structure of the RNA is important. The conformation of the mutant RNAs will be examined both by electron microscopy and with biochemical methods. In addition, this type of analysis will be extended to the conserved central loop region that is described above. It is hoped that the information obtained by our analysis will prove useful not only in understanding the functional significance of structure in coliphage RNAs, but will also help in understanding the role of RNA structure in other genomic viral RNAs and in cellular messenger RNAs.