This project will elucidate the structure and mechanism of Group I self- splicing introns through determination of high resolution crystal structures of tertiary domains which constitute the catalytic core of these ribozymes. Currently, the only crystal structure available for a biologically relevant RNA molecule is of tRNA. Thus, high resolution structural information will be of great interest not only for understanding Group I intron catalysis and evolution, but for understanding RNA structure and function in a variety of biological systems. The Tetrahymena thermophila Group I intron has been chosen for this study because it has been extensively investigated biochemically. Models proposing structural domains within the intron have led to a strategy of crystallizing these regions separately, since they are believed to fold independently and to interact via defined tertiary contacts. The available biochemical data will provide a framework for the interpretation of structural information which is obtained here. Crystal structures will enable the design of rational experiments to understand the molecular basis of the reaction mechanism.
The specific aims of this work are as follows: 1. Solve the crystal structure of the P4P6 tertiary domain from the Tetrahymena intron. This domain of 160 nucleotides contains half of the catalytic core of the intron, including regions that are proposed to be involved in RNA substrate binding. Orthorhombic crystals of the P4-P6 domain have been obtained which diffract to 2.8 Angstroms resolution in our laboratory, and to 2.4 Angstrom resolution at a synchrotron light source. Work is in progress to identify heavy atom derivatives which will be used for phasing. The structure of the P4-P6 molecule will reveal many novel aspects of RNA folding which are required for function. 2. Determine whether the paired regions P3, P7, P8 and P9 (P3-P9) form a second tertiary domain within the catalytic core of the Tetrahymena intron. This portion of the intron contains the remaining conserved nucleotides of the catalytic core, as well as the guanosine binding site. Chemical footprinting will be used to probe its structure in the presence and absence of the P4-P6 domain and the reaction substrates. These experiments will test the current model of Group I intron architecture, and the results will facilitate investigation of the tertiary contacts between the two proposed domains of the intron core. If P3-P9 does form a stable domain on its own, we will begin crystallization trials with appropriate constructs of the RNA. 3. Explore the modes of interaction between the P4-P6 and P3-P9 regions of the Tetrahymena intron. Mutants of both RNAs will be constructed and tested for activity in a reconstitution assay, as well as physically by means of native gel electrophoresis and size exclusion chromatography. The results of these experiments will provide a framework for understanding Group I intron structure and evolution. Our data, together with that of other biochemical laboratories investigating intron structure, will aid in the modeling of interactions between the domains to produce a three- dimensional picture of the complete intron.
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