INTERACTION OF TATA-BINDING PROTEIN WITH UPSTREAM ACTIVATION FACTOR IS REQUIRED FOR ACTIVATED TRANSCRIPTION OF RIBOSOMAL DNA BY RNA-POLYMERASE-I IN SACCHAROMYCES-CEREVISIAE IN-VIVO

Previous in vitro studies have shown that initiation of transcription of ribosomal DNA (rDNA) in the yeast Saccharomyces cerevisiae involves an interaction of upstream activation factor (UAF) with the upstream element of the promoter, forming a stable UAF-template complex; togeth er dth TATA-binding protein (TBP), UAF then recruits an essential fact or, core factor (CF), to the promoter, forming a stable preinitiation complex. TBP interacts with both UAF and CF in vitro. In addition, a s ubunit of UAF, Rrn9p, interacts with TBP in vitro and in the two-hybri d system, suggesting the possible importance of this interaction for U AF function. Using the yeast two-hybrid system, we have identified thr ee mutations in RRN9 that abolish the interaction of Rrn9p with TBP wi thout affecting its interaction with Rrn10p, another subunit of UAF. Y east cells containing any one of these individual mutations, L110S, L2 69P, or L274Q, did not show any growth defects. However, cells contain ing a combination of L110S with one of the other two mutations showed a temperature-sensitive phenotype, and this phenotype was suppressed b y fusing the mutant genes to SPT15, which encodes TBP. In addition, an other mutation (F186S), which disrupts both Rm9p-TBP and Rrn9p-Rrn10p interactions in the two-hybrid system, abolished UAF function in vivo, and this mutational defect was suppressed by fusion of the mutant gen e to SPT15 combined with overexpression of Rrn10p. These experiments d emonstrate that the interaction of UAF with TBP, which is presumably a chieved by the interaction of Rrn9p with TBP, is indeed important for high-level transcription of rDNA by RNA polymerase I in vivo.