Structure of RNA and its Binding Proteins

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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