The long-term goal of this project is to characterize the regulation of gene expression at the translational level. As the applicant's model system, he will investigate the regulation of expression of thymidylate synthase (TS), a critical target in cancer chemotherapy. This enzyme catalyzes the folate-dependent reductive methylation reaction which provides for the sole intracellular de novo source of thymidylate, a key precursor for DNA biosynthesis, and thus, TS plays a central role in maintaining the metabolic requirements of the cell. Previous studies from his laboratory have shown that in addition to its role in catalysis and cellular metabolism, TS also functions as an RNA binding protein. TS binds with high affinity (1-3nM) to two different sites on its own TS mRNA, with one site located in the 5'-untranslated region and the second site in the protein-coding region. This RNA-protein interaction results in the translational repression of TS mRNA with subsequent inhibition of synthesis of new TS protein. Thus, the model of TS translational autoregulation would appear to be biologically relevant in that it offers a rational mechanism for the tight control of TS expression within a given cell. However, treatment of TS protein with inhibitor compounds such as FdUMP or the antifolate inhibitor D1694 alters the normal TS protein-TS mRNA interaction resulting in enhanced translational efficiency of TS mRNA and an increased synthesis of new TS protein. Disruption of this regulatory process might provide an efficient mechanism for malignant cells to protect themselves in response to exposure to cytotoxic stress. To further understanding of the molecular elements underlying the translational regulation of TS, two specific aims are proposed in this project: (1) Characterize the critical cis-acting elements on the TS mRNA that are required for the TS mRNA-TS protein interaction. The applicant plans to identify the essential nucleotide sequences and/or secondary structural elements required for protein recognition of both the 5'-upstream and protein-coding region binding sites, and (2) Characterize the critical trans-acting elements on the TS protein that are necessary for RNA recognition. Specifically, the applicant plans to identify the domain or domains on the TS protein as well as the critical amino acid contact points that mediate the process of RNA binding.
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