We are using a genetic approach to define the role of RNA polymerase II in regulating gene expression. Our experiments have focused on the structure of this multimeric enzyme composed of approximately 12 subunits. Four of these subunits are now cloned in Drosophila and three have been mutated. The vast majority of mutations that exist are in the two largest subunits (215 kd and 140 kd) that constitute greater than 70% of the enzyme's mass. Many of the mutations we have identified in the two largest subunits cause discrete mutant phenotypes suggesting that they are defective in only a subset of the functions or steps required for transcription by RNA polymerase II. The main thrust of our research during the past year has been to begin sequencing the various mutations mentioned above and to ask if they identify specific domains of the proteins that can then be ascribed particular functions by virtue of the mutant phenotypes they elicit. In collaboration with Yan Chen and Arno Greenleaf of Duke University, we have found that mutations that interact with the transcript ion factor, Ubx, cluster in the central region of the largest subunit and the C-terminus of the second largest subunit. The mutation conferring alpha-amanitin resistance in the largest subunit and a putative elongation defective mutation also map to the central region of the largest subunit. Finally, eight mutations in the second largest subunit suppress the lethality of the putative elongation defective mutation in the largest subunit and map to the central region of this subunit. These eight mutations define two genetic groups. Five of the eight are strong suppressors and all change a serine to a cysteine. Three are weak suppressors and map 7 amino acids away, changing a methionine to an isoleucine (2) or valine. This highly conserved region identifies a domain that displays sequence similarity to the disorganized region of bacterial DNA polymerase I and suggests models for the functional and structural relationship of the two largest subunits of RNA polymerase II.
