Synthetic Substrates for Nucleotidyltransferase

Thurlow, David

Abstract

The long term goal of the proposed research is to understand in detail factors that control the catalytic activity of the enzyme ATP,CTP:tRNA nucleotidyltransferase. Since this ubiquitous enzyme is required in eukaryotic cells for biosynthesis of the 3'-terminal CCA sequence of tRNAs, the ability to control its activity may permit regulation of growth in eukaryotic systems, for example in cancerous tissues. The specific goal is to test a model of the tRNA-nucleotidyltransferase interaction in which the enzyme is proposed to extend across the top of the tRNA's L-shaped three dimensional structure, extending from the psi loop, along the psi and acceptor stems, to the 3'-end. The experimental approach is to synthesize synthetic RNA substrates that are structurally similar to the top of a tRNA molecule, and to measure equilibrium (KD) and kinetic (Km and Vmax) constants. Such synthetic substrates have been and will be synthesized using commercially prepared oligodeoxyribonucleotide templates, containing a promoter for phage T7 RNA polymerase and a sequence coding for an RNA stem/loop, similar in sequence and size to the psi loop, psi stem and acceptor stem of a naturally occurring tRNA. These RNAs will be used, together with nucleotidyltransferases from bacterial, fungal and mammalian sources, in nitrocellulose filter binding assays to determine dissociation constants and in enzyme kinetic experiments to determine kinetic constants. Preliminary experiments have established that such RNAs can be synthesized and that they serve as effective substrates for the enzyme. Alterations will be made in the RNA substrates to test effects of changing nucleotides known to be required in chemically unmodified form for the interaction, effects of non Watson-Crick base pairs, effects of increasing or decreasing the distance between the psi loop and the 3'-end, and effects of bulged nucleotides or additional stem/loop structures. From such data, tRNA structural features can be evaluated as to whether they are required for recognition by the enzyme or for stimulation of enzymatic activity. Accordingly, potential inhibitors of this enzyme can be designed to exhibit high affinity and/or an ability to depress rates of the catalytic reaction.