The objective of this research is to elucidate the structure of RNA polymerase III transcription complexes in vivo, and to define the physical properties of the templates in such complexes formed in vitro. Infection of monkey cells by SV-40 viruses containing tRNA genes induces high levels of transcription of those tRNAs. Minichromosomes from such infected cells transcribe the tRNAS in vitro. We will analyze the properties of the transcription complexes on those minichromosomes so as to elucidate their content of transcription factors and to study how viral activator functions such as the adenovirus ElA protein stimulate transcription of tRNA genes. We will measure whether such transcription complexes are incorporated into virions. We will determine whether high salt or detergents can be used to selectively remove chromatin components, while leaving intact the transcription complexes. This may permit determination of the borders of such transcription complexes on the DNA, as well as analyses of the fate of transcription complexes during replication of the template in vivo. To explore the properties of the template within stable transcription complexes, we will assemble transcription complexes on tRNA1met and 5S RNA genes homoduplex and heteroduplex DNAs using Hela cell proteins. We will determine whether complexes assembled on heteroduplex DNAs are transcribed repetitively by DNA polymerase III, or whether the destabilizing effect of the base mismatch interferes with transcription. We will measure the single-stranded character of the template in transcription complexes preceding and following the transit of RNA polymerase III. These studies should provide new information about the stability and expression of genes with Pol III transcription complexes in animal cells.
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