The general objective is to study mechanisms of macro-molecular assembly - in particular, assembly of viruses and of clathrin coated vesicles. It is proposed to extend and complete high-resolution crystallographic structure analyses of two small RNA viruses, tomato bushy stunt virus (TBSV) and turnip crinkle virus (TCV) at 2.5 Angstroms and 3.2 Angstroms resolution respectively. The work on TBSV at 2.5 Angstroms will involve development of software for a new imaging proportional counter (""""""""area detector""""""""). Refinement of TCV structure will require completion of sequence determination of its genomic RNA, by sequencing cloned cDNA. The mechanisms of TCV assembly will be studied in detail. In particular, the structural basis for specific assembly on viral RNA will be investigated by characterization of a salt-stable complex of about 6 coat protein subunits, the 80kDa """"""""minor protein"""""""", and RNA. This complex appears to initiate assembly, and experiments are proposed to demonstrate this function. The RNA participating in this tight interaction will be isolated after partial nuclease digestion, and its sequence will be determined. Its contacts to R or S domains will also be explored. Switching mechanisms important in later stages of assembly appear to involve the disorder/order transition of the subunit arm. Experiments are proposed to test a specific model for potential regulation of subunit conformation. Work on clathrin structure and on its significance for coated vesicle assembly will focus on proteolytic dissection of the molecule into sub-structures. Reassembly properties of clathrin trimers with precisely truncated arms will be studied in order to examine the significance of their distal segments for regulation of cage assembly and design. Attempts will be made to crystallize the globular terminal domain.
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