ANALYSIS OF THE INTERACTION OF THE NOVEL RNA-POLYMERASE-II (POL-II) SUBUNIT HSRPB4 WITH ITS PARTNER HSRPB7 AND WITH POL-II

Under conditions of environmental stress, prokaryotes and lower eukary otes such as the yeast Saccharomyces cerevisiae selectively utilize pa rticular subunits of RNA polymerase II (pol II) to alter transcription to patterns favoring survival. In S. cerevisiae, a complex of two suc h subunits, RPB4 and RPB7, preferentially associates with pol II durin g stationary phase; of these two subunits, RPB4 is specifically requir ed for survival under nonoptimal growth conditions. Previously, we hav e shown that RPB7 possesses an evolutionarily conserved human homolog, hsRPB7, which was capable of partially interacting with RPB4 and the yeast transcriptional apparatus. Using this as a probe in a two-hybrid screen, we have now established that hsRPB4 is also conserved in high er eukaryotes. In contrast to hsRPB7, hsRPB4 has diverged so that it n o longer interacts with yeast RPB7, although it partially complements rpb4(-) phenotypes in yeast. However, hsRPB4 associates strongly and s pecifically with hsRPB7 when expressed in yeast or in mammalian cells and copurifies with intact pol II. hsRPB4 expression in humans paralle ls that of hsRPB7, supporting the idea that the two proteins may posse ss associated functions. Structure-function studies of hsRPB4-hsRPB7 a re used to establish the interaction interface between the two protein s. This identification completes the set of human homologs for RNA pol II subunits defined in yeast and should provide the basis for subsequ ent structural and functional characterization of the pol II holoenzym e.