The objective of this study is to define the manner by which eukaryotic RNA polymerase III recognizes and initiates transcription of tRNA genes. Two approaches will be followed: the first utilizes in vitro manipulation of Xenopus laevis tRNA genes so as to alter their intragenic promoters. Previous studies using bisulfite mutagenesis of X. Laevis tRNAmet tRNA genes have permitted the identification of those G.C pairs within the gene which are important for transcription by pol III. To complete the analysis, techniques for altering A.T base pairs will be developed and applied to the tRNAmet gene. The effect of such alterations upons expression in vivo will be measured. These studies, together with experiments in which putative stem/loop structures in tRNA genes will be manipulated and their effects upon DNA transcriptional activity examined, should provide a detailed picture of the DNA sequence and structural components of a eukaryotic pol III-transcribed gene. The second approach to be utilized involves the construction of recombinant polyoma virus-tRNA DNA's which express tRNAmet at high levels upon introduction into permissive mouse cells. As the chromosomes of these viruses are assembled by cellular proteins and appear to be quite similar, if not identical, to cellular chromatin they permit the study of chromatin structure and composition in isolation from that of the cellular genome. Viral minichromosomes acting as templates for RNA pol III will be prepared and their chromatin organization analyzed. Proteins associated with the minichromosomes will be examined by two dimensional gel electrophoresis. Comparison of recombinant minichromosomes associated with RNA polymerase III, of normal polyoma virus minichromosomes, and of recombinant minichromosomes not transcribed by RNA polymerase III, should reveal which proteins are specifically involved in transcription of tRNA genes. This should complement studies going on in other laboraties in which in vitro systems for tRNA gene transcription are being developed.
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