Program in Macromolecular Structure, Motion, Control

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Biochemical, molecular genetic and high resolution X-ray crystallographic experiments will be used to establish the structural bases for the functional mechanisms of proteins (and enzymes) that interact with DNA or RNA, with a major emphasis on large macromolecular assemblies. Of particular interest are the proteins and nucleic acids involved in facilitating the processes of replication, gene'expression and recombination - the Central Dogma of Molecular Biology. To further illuminate the mechanisms of each step in the process of protein synthesis, we wish 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 complexes whose structures will be pursued include those of the 1) 70S ribosome with bound m-RNA, tRNA and either elongation factor Tu with aminoacyl-tRNA or elongation factor G, 2) the complex of the H. marismortui SOS subunit with fragments of the signal recognition particle, and 3) complexes between either the 70S ribosome or its SOS subunit complexed with the protein secretion and membrane protein insertion channel, the translocon. To explore the differences between the bacterial ribosome and its much large counterpart from eukaryotes, the structures of the 40S subunit, the 60S subunit and/or the 80S ribosome, including an initiation complex formed with IRIS RNA, and using ribosomes isolated from either yeast or rabbit reticulocyte will be pursued. To explore the structural basis of antibiotic specificity, the RNA forming the peptidyl transferase center of H. marismortui 50 S subunit will be replaced by RNA sequences corresponding to those of eubacterial or eukaryotic large subunit rRNA and their complexes with, antibiotics studied. To further understand the mechanism by which the site specific recombinase, gamma-delta resolvase, forms a synaptic complex with two 115 base-pair DNAs and achieves recombination, the structure of the entire 12 subunit complex with DNA will be determined using mutant gamma-delta resolvase. To gain insights into m-RNA splicing the structure of group II intron self-splicing RNA will be pursued. In order to explore the alternate pathway of producing cys-tRNACys used by some archaea, the structure of the archaeal ortholog of Phe-tRNA synthetase that amino acylates tRNACys with phosphoserine will be established with the appropriate substrates bound.