Program in Macromolecular Structure, Motion, Control

Yale University, New Haven CT

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Abstract

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