Biochemical, molecular genetic and high resolution X-ray crystallographic experiments will be used to establish the structural basis for the functioning of proteins and enzymes that interact with DNA or RNA. Establishing the structures of synapsis forming mutants of two homologous recombinases, gammabeta resolvase and GIN, complexed with appropriate DNA substrates will provide insights into site-specific recombination. To understand protein synthesis, we shall establish, in collaboration with Dr. P.B. Moore, the structures of the ribosome and its component subunits both alone and complexed with ligands that are part of the catalytic cycle. The map of the 50S ribosomal subunit from Haloarcula marismortui will be extended to 3A resolution and an atomic model fitted. The structures of its complexes with antibiotic inhibitors and substrate analogous will be analyzed at a similar resolution. The crystal structures of as many archeal 50S ribosomal proteins as possible will be established. The 70S ribosome will be co-crystalized with each of the elongation and termiantion factors as well as tRNA and messenger RNA. The goal is to obtain crystals of the ribosome trapped in as many of the steps in the protein synthesis cycle as possible and to establish their structures at the highest resolution possible. The structural mechanism by which Il3-tRNA synthetase is able to edit out incorrectly activated amino acids and misacylated tRNA will be explored by establishing the crystal structures of the intact enzyme and its functional domains complexed with either structures of the intact enzyme and its function domains complexed with either isoleucyle- or valyl adenalate analogues. To gain insight into the mechanism of a ribozyme and to understand the role of its protein co-factor, mitochondrial tyrosyl-tRNA synthetase form Neurospora complexed with a group I intron RNA will be crystallized and its structure established. In collaboration with Dr. D.M Engelman attempts to solubilize membrane proteins by a variety of methods including mutation will be pursued towards the goal of obtaining high resolution crystal structures.
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