We intend to use biochemical, molecular genetic and high resolution X-ray crystallographic experiments to establish the structural basis for the functioning of proteins and enzymes that interact with DNA and RNA. The structures of proteins involved in both site specific and homologous recombination will be determined in complex with their DNA substrates. Sequence specific recombination will be investigated by establishing the structures of two homologous recombinases, gamma-delta resolvase and GIN, in complex with appropriate DNA substrates. General recombination will be investigated by studying E. coli rec A protein complexed with an ATP analog and single, double and triple-stranded DNA. The endonuclease that cleaves the Holliday junction ruvC, will be studied alone and in combination with a synthetic Holliday junction. The structures of proteins that interact with RNA will be established in a variety of systems. In order to understand the source of specificity of aminoacyl-tRNA synthetase for their amino acid and tRNA substrate, we will investigate complexes of this protein with mutant tRNA molecules as well as substrate analogs of the aminoacyl AMP. The structure of the uncomplexed protein alone will also be established. In order to understand the variety of interactions made by aminoacyl tRNA synthetases and to gain insight into another class of RNA molecules, we shall crystallize and establish the structure of the mitochondrial tyrosyl tRNA synthetase from Neurospora complexed with a group I intron RNA molecule. This synthetase also interacts with tRNA whose complex structure can be determined. Further towards the goal of expanding the structural data base for RNA molecules and their interactions with proteins, we shall determine the structure of the ROM protein complexed with two RNA hairpins that are complementary to each other in a loop region. A nucleolytic fragment of E. coli 5SRNA will also be established at high resolution in order to assess the nature and consequence of non Watson-Crick base-pairs to this RNA structure. Towards the long-term goal of providing a structural basis for understanding protein synthesis on the ribosome, we shall work to establish the structure of the elongation factor G complexed with a GTP analog. Initial investigations of crystallization and structural work on the ribosome will be made.
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