We are analyzing the mechanism of transcription by eukaryotic RNA polymerase III. We have focused on the polymerase III transcription machinery from the silkworm, Bombyx mori, because there is evidence for tissue-specific regulation of polymerase III activity in this organism. This is a fundamental biological problem. All eukaryotes require the products of correct transcription by polymerase III. Moreover, since it is likely that there are mechanistic features common to all three eukaryotic RNA polymerases, analysis of any one enzyme should also give insight into the others. Using in vitro mutagenesis we have already defined the boundaries of the cisacting elements that direct polymerase III transcription of a silkworm tRNAA1a gene. We have found that the region required for full transcriptional activity of this gene is remarkably large. It includes the coding region, and extends beyond it both upstream and downstream. In the proposed work we will determine whether such large control elements are general features of polymerase III templates, and we will identify the functions they provide. The functional analyses we propose take advantage of our ability to manipulate the components of the Bombyx polymerase III transcription machinery that we have resolved. By varying the elements that act in cis, as well as those that act in trans, we will determine how interactions between these elements lead to correct transcription by polymerase III.
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