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 domains in the two largest subunits interact with each other?", we have identified numerous extragenic suppressor mutations that correct mutant phenotypes elicited by pol II mutations. Eight mutations in the second-largest subunit of pol II were recovered as suppressors of RpII215-K1. Five of these mutations are strong, restoring viability to that of wild-type flies, and resulting from a serine to cysteine substitution at amino acid 675, while three are mild, restoring viability to half of that of wild-type flies and resulting from a methionine to isoleucine or valine substitution at amino acid 683. Two of three intragenic suppressors are in the same domain as the original RpII215-K1 mutation; however, none of the intragenic suppressors were precise revertants. We examined 39 alleles in the largest subunit for interactions with the suppressors. We identified the recessive-lethal allele, RpII215-120, as being suppressed by the strong suppressors. Sequence analysis identified the change responsible for RpII215-120 as being in the same domain as RpII215-K1 and two of the intragenic suppressors. We predict that these domains directly interact with each other.
NIH ExPorter
NIH ExPorter