Transcription of chromosomal rRNA genes by both RNA polymerase I and II in yeast uaf30 mutants lacking the 30 kDa subunit of transcription factor UAF.

UAF, a yeast RNA polymerase I transcription factor, contains Rrn5p, Rrn9p, Rrn10p, histones H3 and H4, and uncharacterized protein p30. Mutants defective in RRN5, RRN9 or RRN10 are unable to transcribe rDNA by polymerase I and grow extremely slowly, but give rise to variants able to grow by transcribing chromosomal rDNA by polymerase II. Thus, UAF functions as both an activator of polymerase I and a silencer of polymerase II for rDNA transcription. We have now identified the gene for subunit p30. This gene, UAF30, is not essential for growth, but its deletion decreases the cellular growth rate. Remarkably, the deletion mutants use both polymerase I and II for rDNA transcription, indicating that the silencer function of UAF is impaired, even though rDNA transcription by polymerase I is still occurring. A UAF complex isolated from the uaf30 deletion mutant was found to retain the in vitro polymerase I activator function to a large extent. Thus, Uaf30p plays only a minor role in its activator function. Possible reasons for slow growth caused by uaf30 mutations are discussed.

Yeast RNA polymerase I enhancer is dispensable for transcription of the chromosomal rRNA gene and cell growth, and its apparent transcription enhancement from ectopic promoters requires Fob1 protein.

At the end of the 35S rRNA gene within ribosomal DNA (rDNA) repeats in Saccharomyces cerevisiae lies an enhancer that has been shown to greatly stimulate rDNA transcription in ectopic reporter systems. We found, however, that the enhancer is not necessary for normal levels of rRNA synthesis from chromosomal rDNA or for cell growth. Yeast strains which have the entire enhancer from rDNA deleted did not show any defects in growth or rRNA synthesis. We found that the stimulatory activity of the enhancer for ectopic reporters is not observed in cells with disrupted nucleolar structures, suggesting that reporter genes are in general poorly accessible to RNA polymerase I (Pol I) machinery in the nucleolus and that the enhancer improves accessibility. We also found that a fob1 mutation abolishes transcription from the enhancer-dependent rDNA promoter integrated at the HIS4 locus without any effect on transcription from chromosomal rDNA. FOB1 is required for recombination hot spot (HOT1) activity, which also requires the enhancer region, and for recombination within rDNA repeats. We suggest that Fob1 protein stimulates interactions between rDNA repeats through the enhancer region, thus helping ectopic rDNA promoters to recruit the Pol I machinery normally present in the nucleolus.