Structure of RNA and its Binding Proteins

摘要丰富的MCB 98-08127 1.通过对核糖核酸的三维结构的了解，我们对其生物学功能的理解有了很大的提高。本研究的总体目标是通过单晶X射线结晶学确定各种RNA的三维结构基序。由于RNA的生物活性通常是通过与其相互作用的蛋白质来表达的，因此本研究旨在解决与RNA结合的RNA结合蛋白的结构问题。遗传信息流涉及到信使核糖核酸的产生和随后转化为蛋白质。这一过程通常会被翻译监管机构修改。在噬菌体T4的生产中，蛋白RegA是一种翻译调节因子，可以改变35mRNAs中的蛋白质合成。该蛋白质的三维结构已被解决，本研究旨在使该蛋白质与其一个或多个RNA底物共结晶，以了解信使选择的机制。一个重要的RNA构象基序是RNA链以特定方式折叠回自身的假结。RNA假结改变了mRNA的阅读框架，是许多病毒感染的重要组成部分。甜菜西部黄化病毒RNA伪结节的结构已经在P.I.S实验室中解决了。本研究的实验考察了它的三维结构是否提供了关于这种RNA构象如何在翻译中改变阅读框架的见解。编辑酶双链RNA腺苷脱氨酶具有多个RNA结合域，以及左撇子Z-DNA的结合域。本研究旨在表达RNA结合域，并将其与双链RNA底物共结晶。这一结果可能为深入了解该酶的编辑机制提供依据。痘苗病毒编码的E3L蛋白还含有一个双链RNA结合基序和一个Z-DNA结合基序。其与核酸结合成分的晶体结构的解决方案可能提供对其作用模式的洞察。摘要丰富的MCB 98-08127 2.生物系统中的非技术信息主要编码在基因组的dna中。DNA片段被用来指导RNA的合成，而RNA在指导蛋白质合成方面起着积极的作用。然而，RNA本身在监测和改变遗传信息的表达方式方面具有可考虑的影响。为了了解这是如何发生的，这项研究的一个总体目标是使用单晶X射线分析来确定RNA的三维结构。一旦知道了RNA的三维结构，就可以更好地理解它在生物系统中的作用。遗传信息流涉及RNA的生产和随后翻译成蛋白质。然而，这一过程通常会被一个调节器改变，即一种蛋白质，它与指导蛋白质合成的RNA结合。这项研究涉及在T4噬菌体中发现的系统，在该系统中，一种名为RegA的蛋白质与信使RNA的选择性区域结合并调节其翻译。早些时候已经解决了RegA蛋白的三维结构，这使得人们可以通过与其结合的信使RNA片段来研究其复合体的结构。正是这种结合作用的性质决定了合成多少蛋白质。RNA决定蛋白质生成量的另一个例子是RNA折叠成一种称为假结的结构。也就是说，该结构是在双链区域折叠的，但它不是一个真正的结，因此是伪结。假结作用于翻译系统，调节形成的蛋白质数量。还解决了用X射线衍射分析从甜菜西黄病毒中获得的RNA假结节的三维结构。这种结构信息被用来研究假结如何在修饰蛋白质合成中起作用。另一种RNA修饰可以在与双链RNA结合的酶中看到，并改变RNA中核苷酸的化学性质。这被称为编辑酶，因为它改变了信使RNA的一个组成部分，而信使RNA反过来又改变了蛋白质中特定氨基酸的身份。因此，这项研究的另一个目的是了解这种编辑酶是如何工作的，以及它如何与双链RNA结合来进行这种编辑活动。为了达到这个目的，这种酶的片段将被结晶，并在与双链RNA结合时解决它们的结构问题，这样就可以确定编辑过程的机制。这项研究的总主题是了解RNA的三维结构及其与蛋白质的相互作用如何改变生物系统中的遗传信息流，从而导致蛋白质合成方式的改变。2.

Abstract Rich MCB 98-08127 1. Technical Our understanding of the biological function of RNA is enhanced considerably by knowledge of its three-dimensional structure. The general objective of this study is to determine various RNA three-dimensional structural motifs by single crystal X-ray crystallography. Since the biological activity of RNA is often expressed through the proteins that it interacts with, this study aims at solving the structure of RNA binding proteins in conjunction with the RNA. The flow of genetic information involves the production of mRNA and its subsequent translation into protein. That process is often modified by a translational regulator. In bacteriophage T4 production, the protein regA is a translational regulator that modifies protein synthesis in 35 mRNAs. The three-dimensional structure of that protein has been solved, and this study is to co-crystallize the protein with one or more of its RNA substrates in order to understand the mechanism of messenger selection. An important RNA conformational motif is the pseudoknot in which the RNA strand folds back on itself in a specific manner. The RNA pseudoknot changes the mRNA reading frame and is an important component of many viral infections. The structure of the RNA pseudoknot from the Beet Western Yellow Virus has been solved in the P.I.'s lab previously. Experiments in this study examine if its three-dimensional structure provides insight into how this RNA conformation acts to change the reading frame in translation. The editing enzyme double-stranded RNA adenosine deaminase has several RNA binding domains, as well as a binding domain for left-handed Z-DNA. This study is to express the RNA binding domains and co-crystallize them with double-stranded RNA substrates. The results may provide insight into the editing mechanism of this enzyme. The E3L protein encoded by Vaccinia virus also contains a double-stranded RNA binding motif as well as a Z-DNA binding motif. Solution of its crystal structure w ith nucleic acid binding components may provide insight into its mode of action. Abstract Rich MCB 98-08127 2. Non-technical Information in biological systems is encoded primarily in the DNA of the genome. Segments of the DNA are used to direct the synthesis of RNA which is active in directing the synthesis of proteins. However, RNA itself has a consider-able impact in monitoring and changing the way in which genetic information is expressed. In order to understand how this occurs, a general objective of this study is the determination of the three-dimensional structure of RNA using single-crystal X-ray analysis. Once the three-dimensional structure of RNA is known, a better understanding of how it acts in biological systems can be achieved. The flow of genetic information involves the production of RNA and its subsequent translation into proteins. However, that process is often modified by a regulator, that is a protein that binds to the RNA that directs protein synthesis. This study concerns the system that is found in the bacteriophage T4 in which a protein called regA binds to selective regions of messenger RNA and regulates its translation. The three-dimensional structure of the regA protein was earlier solved, which allows pursuing the structure of its complex with segments of messenger RNA to which it binds. It is the nature of this binding interaction that determines how much protein is synthesized. Another example in which RNA determines the amount of protein formed is found in the folding of RNA into a structure which is called a pseudoknot. That is, the structure is folded in double-stranded regions, but it is not a genuine knot, hence pseudoknot. The pseudoknot acts on the translational system and regulates the amount of protein that is formed. The three-dimensional structure of the RNA pseudoknot obtained from a Beet Western Yellow Virus using X-ray diffraction analysis was also solved. This structure information is used to study how a pseudoknot acts i n modifying protein synthesis. Another RNA modification may be seen in an enzyme which binds to double-stranded RNA and changes the chemical nature of the nucleotides in the RNA. This is called an editing enzyme because it changes a component of the messenger RNA which, in turn, changes the identity of a particular amino acid in a protein. Another aim of this study is thus to understand how this editing enzyme works and how it binds to double-stranded RNA to carry out this editing activity. Toward this end segments of this enzyme are to be crystallized and solved for their structures when bound to double-stranded RNA, so that the mechanics of the editing process can be determined. The general theme of this research is to understand how the three-dimensional structure of RNA and its interaction with proteins changes the flow of genetic information in biological systems, resulting in modifications in the way proteins are made. 2