The overall objective of this work is to understand the specificity of the mechanisms that affect the regulation and maturation of eukaryotic RNA transcripts. In particular, we shall construct a variety of hybrid genes in which RNA polymerase I promoter sequences are fused to the body of the gene coding for the bacterial chloramphenicol transferase (CAT) protein. Following transfection into cultured mammalian cells, we shall assay at both the RNA and protein levels for the transient expression of these hybrid genes which have been constructed to contain SV40 splice and polyadenylation sites 3' to the CAT coding region. Using a variety of techniques, we shall determine whether the transcripts of the resultant genes are accurately initiated under the direction of the polymerase I promoter sequences, whether the nascent hybrid transcripts are capped, polyadenlyated, and spliced as they would be if initiated by RNA polymerase II, whether the RNAs are transported to the cytoplasm and whether the final RNA products are utilized for translation of functional protein. Furthermore, using this fusion gene protocol we shall determine whether polymerase II enhancer sequences can augment transcription from polymerase I promoters and we will investigate the characteristics of such enhancement. With these experiments, we will begin to test some current models of how enhancer sequences function to stimulate transcriptional activity. The generality of these results will also be examined through a similar series of experiments which will test whether a functional, processed protein encoding RNA can be transcribed by polymerase III from a polymerase III promoter sequence linked to a protein coding gene and whether transcription from such a gene is up-modulated by a polymerase II enhancer sequence.
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