Transcription Factor UAF, Expansion and Contraction of Ribosomal DNA (rDNA) Repeats, and RNA Polymerase Switch in Transcription of Yeast rDNA

Abstract

Strains of the yeast Saccharomyces cerevisiae defective in transcription factor UAF give rise to variants able to grow by transcribing endogenous ribosomal DNA (rDNA) by RNA polymerase II (Pol II). We have demonstrated that the switch to growth using the Pol II system consists of two steps: a mutational alteration in UAF and an expansion of chromosomal rDNA repeats. The first step, a single mutation in UAF, is sufficient to allow Pol II transcription of rDNA. In contrast to UAF mutations, mutations in Pol I or other Pol I transcription factors can not independently lead to Pol II transcription of rDNA, suggesting a specific role of UAF in preventing polymerase switch. The second step, expansion of chromosomal rDNA repeats to levels severalfold higher than the wild type, is required for efficient cell growth. Mutations in genes that affect recombination within the rDNA repeats, fob1 and sir2, decrease and increase, respectively, the frequency of switching to growth using Pol II, indicating that increased rDNA copy number is a cause rather than a consequence of the switch. Finally, we show that the switch to the Pol II system is accompanied by a striking alteration in the localization and morphology of the nucleolus. The altered state that uses Pol II for rDNA transcription is semistable and heritable through mitosis and meiosis. We discuss the significance of these observations in relation to the plasticity of rDNA tandem repeats and nucleolar structures as well as evolution of the Pol I machinery.All eukaryotic cells have three distinct RNA polymerases that normally transcribe different sets of nuclear genes. RNA polymerase I (Pol I) is unique in that in most eukaryotic organisms, its sole function is the transcription of genes for large rRNAs (rDNA). Dedication of a separate RNA polymerase to rDNA transcription is a feature unique to eukaryotes and must have had strong selective advantages for eukaryotic organisms in evolution. However, we have recently discovered that mutants of the yeast Saccharomyces cerevisiae which are defective in transcription factor UAF (upstream activation factor) give rise to variants which now grow by transcribing endogenous rDNA by RNA polymerase II (Pol II) (39). (In this paper, we use the term transcription of rDNA to imply transcription of the gene encoding the 35S precursor rRNA, although the 5S RNA gene transcribed by RNA polymerase III is a part of the rDNA repeat unit in S. cerevisiae.) Thus, yeast cells have an inherent ability to use Pol II for rDNA transcription, but this transcription activity is apparently silenced in normal cells. Studies of the processes which enable yeast cells to grow without using the Pol I machinery may be important for understanding the normal Pol I machinery and for gaining insight into the significance of its evolution. In this paper, we describe our finding that the switch to growth using the Pol II system consists of two steps; the first step is a mutational alteration in UAF, and the second step is an expansion of chromosomal rDNA repeats. Th...

Sir2 Represses Endogenous Polymerase II Transcription Units in the Ribosomal DNA Nontranscribed Spacer

“…Unlike the Arabidopsis system, in S. cerevisiae, transcription by Pol I is not increased in cells that lack the histone deacetylase RPD3 (Oakes et al, 1999(Oakes et al, , 2006. Likewise, no changes in Pol I transcription have been observed in cells lacking Sir2 (Oakes et al, 1999(Oakes et al, , 2006. Despite the differences, a common thread between the yeast and plant systems is that loss of specific HDACs results in rDNA that is more like euchromatin, and, subsequently, has altered function.…”

“…Previous studies have characterized mutants that lack subunits of Pol I and survive by transcribing the 35S rRNA genes with Pol II (Conrad-Webb and Butow, 1995; Vu et al, 1999). However, in RNA from sir2⌬ cells, Pol II-derived 35S rRNA transcripts have not been detected (Oakes et al, 1999(Oakes et al, , 2006. Moreover, we did not detect high levels of K4-trimethylated H3 or Pol II over the 35S rRNA gene, which would be expected if the 35S rRNA gene was being transcribed by Pol II.…”

“…In several Arabidopsis species, disruption of the histone deacetylase (HDAC) activities of HDT1, a plant-specific HDAC, or HDA6, a member of the RPD3 family of HDACs, caused normally silenced rDNA repeats to acquire characteristics of active rDNA repeats, including high levels of Pol I and K4-methylated H3, and low levels of K9-methylated H3 and DNA methylation (Lawrence et al, 2004;Probst et al, 2004;Earley et al, 2006). Unlike the Arabidopsis system, in S. cerevisiae, transcription by Pol I is not increased in cells that lack the histone deacetylase RPD3 (Oakes et al, 1999(Oakes et al, , 2006. Likewise, no changes in Pol I transcription have been observed in cells lacking Sir2 (Oakes et al, 1999(Oakes et al, , 2006.…”

RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing

“…Quantification of rDNA copy number was performed as described (43), except that Southern blot hybridization of the rDNA probe was normalized to the URA3 gene probe. Sucrose gradient centrifugation analysis of polysome profiles was performed essentially as described (44).…”

Role of TATA Binding Protein (TBP) in Yeast Ribosomal DNA Transcription by RNA Polymerase I: Defects in the Dual Functions of Transcription Factor UAF Cannot Be Suppressed by TBP

“…Thus, they concluded that high levels of TBP can bypass the requirement for UAF and that TBP directly stimulates transcription from the core transcriptional machinery (1). We found previously that mutations in UAF components immediately lead to a polymerase switch in rRNA transcription from Pol I to Pol II (17,20). This state is called N-PSW.…”