The aim of the proposed work is to investigate the mechanism of transcription by silkworm RNA polymerase III in vitro. Much of our effort will focus on a novel transcription component, Factor X, that our laboratory has recently isolated. This factor is an essential part of the machinery that transcribes eukaryotic tRNA and 5S genes. It is generally assumed that such machinery is composed entirely of polypeptides. The remarkable feature of Factor X is that it appears to be an RNA rather than a protein. The finding of an RNA with transcription activity is unprecedented. The discovery of Factor X came from our systematic fractionation of the components required for tRNA and 5S RNA transcription in vitro. It is distinct from the components previously resolved by our laboratory and others: polymerase III, and the transcription factors A, B, C, and D. Factor X appears to be a nucleic acid because it is resistant to heat, detergent, phenol, and protease, but sensitive to nuclease. It appears to be an RNA because it is resistant to DNAse, but sensitive to RNAse and alkali.
The specific aims of this proposal are: (1) to identify the critical segments of the large tRNA(Ala)C promoter that are essential for function, (2) to purify Factor X, as well as the other silkworm transcription factors in order to establish their identities unambiguously, (3) to assign specific functions to Factor X and the other transcription factors by (a) determining whether they participate in factor-template and factor-factor interactions, (b) determining which phase of the cell cycle requires Factor X, and (c) constructing mutant versions of Factor X to test specific hypotheses for its function.
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