The goal of this work is to discover the mechanism of differential transcription in vitro of two kinds of tRNA genes. These genes encode alanine tRNA in the silkworm, Bombyx mori, and they differentially regulated in this organism. One class (tRNAAlaC genes) encodes alanine tRNA that is common to all silkworm cell types. The other class (tRNAAlaSG genes) encodes a distinct alanine tRNA that is found only the silkgland. The proposed study addresses the general problem of eukaryotic gene control, and the specific problem of transcription by RNA polymerase III. Since RNA polymerase III acts in all cells of all eukaryotes, understanding its function has broad biological and medical significance. The transcription properties displayed by tRNAAlaSG genes in vitro are very different from those displayed by tRNAAlaC genes -- and probably provide the basis for differential regulation in vivo. Therefore, a mechanistic analysis of these in vitro properties is biologically relevant. The interesting features of tRNAAlaSG transcription in vitro are 1.) It is extremely inefficient (relative to tRNAAlaC transcription) under standard conditions, and 2.) It is as efficient as tRNAAlaC transcription under special conditions. These two states could correspond to the inactivity of tRNAAlaSG genes in most silkworm cell types, and their high level of activity (equivalent to tRNAAlaC genes) in the silkgland. The molecular basis of both of these properties will be investigated. Specifically, the proposed experiments will identify the interaction(s) with the transcription machinery, and the step in the transduction cycle that is less efficient for tRNAAlaSG templates. When the defect in tRNAAlaSG transcription has been localized, the mechanism for overcoming it will be analyzed. To determine whether specific stimulation of tRNAAlaSG transcription in vitro is due to a qualitative or a quantitative change in the transcription machinery, the stimulatory component(s) will be resolved from the remainder of the transcription machinery, and tested for its specific activity on the two kinds of tRNAAla genes.
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