The major transcriptional activities in eukaryotic cells are devoted to the synthesis of rRNAs and tRNAs by RNA polymerases I and III. The in vitro transcription systems derived for Pol I and III, compared to those for Pol II, are efficient, specified by fewer proteins, and more often mimic the transcription events observed in vivo. We have made substantial progress in purification and characterization of the Drosophila Pol I and Pol III transcription components. The species specific nature of these components and the unique features of these Drosophila transcription systems have led us to examine the role of these proteins in specific regulatory effects. Our results are the first to show that serum, TPA (A tumor-promoting phorbol ester), and a calcium ionophore can up-regulate rRNA and tRNA gene expression in Drosophila. Furthermore, we have been able to derive cell extracts that reproduce this transcription increase in vitro. Using transcription reconstitution assays and partially purified protein factors, we will determine the relative activity levels of Pol I, TFIC, TFID, and Pol III, TFIIIB, and TFIIIC in non-stimulated and stimulated cell extracts. Stable transcription formation and gel shift assays will be used to analyze alterations in the DNA binding functions of TFID and TFIIIC. These results will define, for the first time, Pol I and III regulatory events mediated by serum, TPA and calcium ionophore, and the relationships among these events. Since the induction of specific genes by phorbol esters in Drosophila has not been previously demonstrated, our studies will provide new insights as to its mechanism for gene regulation in this system. Once we have defined which of the factors are involved in these regulatory processes, we will further determine the structural modifications in specific polypeptides which give rise to their altered activities. Qualitative and quantitative differences in the polypeptides which are shared by Pol I and III, and those polypeptides which comprise the factor subunits will be examined by immunological approaches. We will use both currently available antibodies and antibodies derived from purified Drosophila components. We will expand our purification protocols to more completely purify these components, focusing on those proteins which we find to be altered in the regulatory events. In total, these experiments can offer unique insights into gene regulation in Drosophila, into the control of Pol I and III gene expression, and into potential novel mechanisms of the co-regulation of these two systems.
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