INTEGRATION SPECIFICITY OF THE TY3 RETROTRANSPOSON

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Ty3 is a gypsy-like retrotransposon in Saccharomyces that integrates close to the site of polymerase III transcription initiation. Ty3 is 5.4 kbp in length and is composed of long terminal repeats of 340 bp flanking an internal domain. It is transcribed into a 5.2kb, polyadenylated RNA which is analogous to the genomic RNA of retroviruses. GAG3 and POL3 reading frames encode the structural proteins--capsid and nucleocapsid--of a 50 nm nucleoprotein particle and catalytic proteins--protease, reverse transcriptase, and integrase (IN)- -required for processing, replication and integration of the element. Ty3 organization and proteins resemble those of animal retroviruses. Retroviruses do not integrate randomly in vivo, yet neither host nor retroviral proteins, other than IN, have been identified as present at the integration site. The long-term objective of this project is to exploit the molecular genetic approaches available in yeast and the relatively defined position specificity of Ty3 to investigate the mechanism of delivery of a retroid element to an integration site: 1) An early immediate objective is to document physical changes in IN over a timecourse, and to determine how they relate to integration. These changes include phosphorylation and proteolytic processing. 2) Host proteins involved in integration will be identified in GAL4 hybrid screen for proteins which interact with IN and by cloning and characterization of genes which complement host mutants for Ty3 integration. 3) Studies of retroviral and Ty3 insertion suggest that genomic context affects integration. The 5S RNA gene carried on a plasmid, but not in its native chromosomal context, is a target for Ty3 transposition. Yeast mutants in nucleolar structure, supercoiling, and chromatin will be examined to determine whether genomic exclusion can be assigned to the nuclear environment of the 5S gene. 4)In vitro, retroviral IN is sufficient for random integration of molecules resembling the replicated genome into naked DNA, but in vivo, it is not known whether IN is in the core particle or if it acts independently during integration. Ty3 IN will be expressed under a heterologous promoter, alone and in fusions to Ty3 particle-targeting and nuclear- targeting sequences, and its activity will be tested by genetic and physical means. Activity would not only indicate a degree of IN autonomy in the cell, but would provide an indispensable tool for future studies of integration distinct from replication. 5) An in vitro integration system modeled on the retrovirus system, but incorporating host proteins, will be developed in order to identify the interactions responsible for docking the Ty3 integration complex at the target. 6) Ty3 IN mutants will be constructed and selected in order to identify domains responsible for interaction with the termini of the integration precursor (conserved in all retroid elements) and domains that affect integration specificity. The ability of a heterologous DNA binding domain to confer novel insertion specificity will also be examined. These experiments in a simple eukaryote should lead to the development of models which can be explicitly tested in retroviral systems.