Maintenance of the tRNA 3' end

Thomas Jefferson University, Philadelphia PA

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Abstract

DESCRIPTION (provided by applicant): All transfer RNA (tRNA) molecules contain a CCA sequence at the 3' end that is the site of amino acid attachment during decoding of genetic information, and is the site of initiation of primer extension during replication of retroviruses. The CCA sequence is also implicated in the evolution and development of bacterial pathogenesis. The synthesis and maintenance of the CCA sequence is catalyzed by the CCA enzyme (ATP (CTP): tRNA nucleotidyl transferase), which is present in every living organism. The nature of CCA synthesis is of immense interest, because its specificity is achieved without a nucleic acid template. To elucidate the molecular basis of the specificity of CCA, the long-term goal is to address how the enzyme recognizes CTP from ATP and vice versa at each step of addition, whether the enzyme has an induced-fit conformational change after nucleotidebinding, and whether the induced-fit is the rate-limiting step. These questions are the root cause of the fidelity, which provides a mechanism to relate the template-independent CCA enzyme to template-dependent polymerases. As a contribution to the long-term goal, this proposal will address the specificity of nucleotide-binding, induced-fit mechanisms, and the specific role of ATP in controlling the specificity. Preliminary studies have shown that, in the absence of ATP, the CCA enzyme will mis-incorporate and synthesize poly(C). The role of ATP in manifestation of specificity is a distinct feature of the CCA enzyme. Further, preliminary studies have also identified a 3' to 5' hydrolytic activity that is reminiscent of the editing or proof-reading activity of DNA polymerases. This activity will be characterized to gain better insights into its role in the specificity of CCA. Because of the importance of CCA in maintenance of cellular life, the proposed studies will have a strong impact on human health and diseases.