The objectives of this proposal are (1) to identify sequence and/or structural features in prokaryotic initiator tRNAs which specify their distinctive properties, (2) to understand the molecular mechanisms by which various components of the protein synthetic machinery distinguish initiator tRNAs from other tRNAs and (3) to generate animal cell lines and Drosophila strains carrying inducible suppressor tRNA genes. The approach is to generate mutant tRNAs and analyze their function at each of the steps on the pathway to initiation in vitro and in vivo. The role of two of the unique features of initiator tRNAs have been identified.
We aim to identify the role of the third feature. Concurrently, attempts to convert an elongator tRNA into an efficient initiator by introducing sequence features found in initiator tRNA will continue. Mutants defective at specific steps in initiation have been obtained. Some are inactive because they cannot be formylated, others because they cannot bind to the P site on the ribosome. We will investigate whether overexpression or mutation of any E. coli gene will suppress these defects. We will identify the genes involved and analyze the mode of suppression. this work could lead to identification of components which interact with initiator tRNA and an understanding of how such interactions contribute to protein synthesis. It may also lead to identification of new genes. Studies on underlying molecular mechanisms in the highly specific recognition of initiator tRNA by Met-tRNA transformylase will continue. The key elements for this appear to be clustered in the acceptor stem, a mismatch or a weak base pair at the end of the acceptor stem being important. We will investigate these structural requirements. We will probe the topology of enzyme .tRNA interaction in solution and attempt to crystallize the enzyme .tRNA complex. Such work should be facilitated by a method we have developed for large scale preparation of analogues of methionyl-tRNA in which methionine is linked to tRNA through a stable amide bond. Finally, we propose to use current knowledge of tRNAs and aminoacyl-tRNA synthetases to regulate expression of suppressor tRNA genes or their function in eukaryotic cells. The goal is to develop methods that are generally applicable for generating mammalian cell lines and Drosophila strains carrying inducible suppressors.
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