This grant proposal deals entirely with a newly described property shared by TAg, the transforming product of the oncogenic virus SV40, and the oncogene product p53. TAg and p53 each has an RNA-bound proteolytic peptide which elutes heterogenously from HPLC due to varying lengths of bound RNA. Through enzymatic manipulation of the oligonucleotide-bound peptides, sequence determination of the peptides, and characterization of the bonds (largely finished) we have established unambiguously that the two proteins are bound covalently to RNA. The task now is to determine what part this RNA-protein bond plays in the biological activities of TAg in lytic and transforming infections. In order to establish the biological significance of the covalently bound RNA to TAg function, we propose to map the Ser-CMP bond to the precise serine residue of TAg by amino acid sequencing. A number of arguments are advanced that indicate the peptide is from near the carboxyl terminus of TAg. The serine residue will be altered by site-directed mutagenesis in order to determine whether lytic growth, transformation, or the adenovirus-helper function are affected. The single most significant question at this point is, What is the RNA? Identification of the Tag-and p53-bound RNAs will allow us to make reasonable hypotheses about the biochemical function of the protein-RNA complexes and the feasibility of a postulated concerted reaction. Because TAg and p53 form a tight protein- protein complex, binding of RNA by each suggests they may be carrying out a concerted reaction involving the RNA. The effects of the RNA upon the sedimentation and density of the TAg-p53 complex will be determined. We postulate that the RNA-bound complex can be gently purified because of its postulated high S value and density. After sequencing Tl oligonucleotides from the bound RNA, small complementory DNA fragments will be synthesized for use as probes to identify intact RNA in the purified TAg-p53-RNA complex. Parameters of the putative concerted RNA binding reaction will be studied, such as turnover of the RNA, site occupation and in vitro binding. Our hypothesis is that the TAg-p53 complex functions in RNA processing or transport in some manner, explaining the ability of TAg to modulate host gene expression and transform the cell. The long term goal is to elucidate the mechanism of this effect on host gene expression.
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