Work with yeast shows that the core RNA polymerase II (pol II) protein machinery consists of twelve subunits and the holoenzyme possesses over 50. Multicellular organisms must regulate gene expression as in yeast but also integrated it through a developmental program and among many cell types and tissues. Biochemical approaches have identified many of the factors that communicate with the basal machinery; however, we are ignorant of the mechanism by which pol II integrates these signals in higher organisms. Drosophila is the most amenable multicellular organism for detailed genetic analyses to dissect the functional interactions of these factors. Our approach is to isolate mutations in genes (extragenic) that enhance or suppress mutant phenotypes elicited by specific pol II alleles of the two largest subunits, encoded by the RpII215 and RpII140 loci. The interacting genes identified by this classical and molecular genetic approach are further analyzed using in vitro transcription assays of nuclear extracts from mutant flies and by transforming and/or transfecting cell lines with mutant genes and reporter constructs. Understanding the molecular basis of these interactions will elucidate the mechanism by which the transcriptional machinery plays a role in gene regulation. To answer the question """"""""Which proteins interact with RNA polymerase II?"""""""", we have identified numerous extragenic enhancers of mutant phenotypes elicited by pol II mutations. The best characterized of these is an interaction with the transcription factor Ubx. We have focused on the determination of the mechanism of this interaction or enhancement. Our results suggest that Ubx protein signaling to mutant pol II is abnormal resulting in the mis-expression of Ubx target genes. We directly tested this hypothesis using nuclear extracts from mutant flies in Ubx dependent in vitro transcription assays. We show that nuclear extract from RpII2154 flies responds more slowly to Ubx than does wild-type extract. Furthermore, in mixtures of the two extracts, the extract from mutant flies interferes with the response of wild-type extract. These results parallel observations of mutant phenotypes seen in adult flies and suggest there are changes in RNA polymerase II structure following signals from transcription factors.
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