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

Abstract

Initiation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae involves upstream activation factor (UAF), core factor, the TATA binding protein (TBP), and Rrn3p in addition to Pol I. We found previously that yeast strains carrying deletions in the UAF component RRN9 switch completely to the use of Pol II for rRNA transcription, with no residual Pol I transcription. These polymerase-switched strains initially grow very slowly, but subsequent expansion in the number of rDNA repeats on chromosome XII leads to better growth. Recently, it was reported that TBP overexpression could bypass the requirement of UAF for Pol I transcription in vivo, producing nearly wild-type levels of growth in UAF mutant strains (P. Aprikian, B. Moorefield, and R. H. Reeder, Mol. Cell. Biol. 20:5269-5275, 2000). Here, we demonstrate that deletions in the UAF component RRN5, RRN9, or RRN10 lead to Pol II transcription of rDNA. TBP overexpression does not suppress UAF mutation, and these strains continue to use Pol II for rRNA transcription. We do not find evidence for even low levels of Pol I transcription in UAF mutant strains carrying overexpressed TBP. In diploid strains lacking both copies of the UAF component RRN9, Pol II transcription of rDNA is more strongly repressed than in haploid strains but TBP overexpression still fails to activate Pol I. These results emphasize that UAF plays an essential role in activation of Pol I transcription and silencing of Pol II transcription of rDNA and that TBP functions to recruit the Pol I machinery in a manner completely dependent on UAF.

FIG. 1

Polymerase switch phenotype in rrn5Δ, rrn9Δ, and rrn10Δ strains. Diploid strains NOY679 (RRN5/rrn5Δ), NOY678 (RRN9/rrn9Δ), and NOY677 (RRN10/rrn10Δ) were transformed with the GAL7-35S rDNA plasmid pNOY199 and sporulated, and then tetrads were dissected on galactose plates. Tetrads corresponding to rrn5Δ, rrn9Δ, and rrn10Δ (as indicated by slow growth) were restreaked once onto another galactose plate (lacking tryptophan, to maintain pNOY199), and then aliquots of 10-fold serial dilutions of two independent colonies from these strains were spot tested on YEP-galactose (Gal) and YEPD (Glu) plates, as shown. Plates were incubated for 6 days at 30°C.

FIG. 2

Overexpression of TBP in an rrn9Δ strain carrying a 2μm (2μ)-TBP plasmid (pNOY419). Protein extracts prepared from rrn9Δ N-PSW strains carrying the vector control (NOY1001) or carrying 2μm-TBP (NOY1000) were analyzed by Western blotting using affinity-purified TBP antibody. Ten micrograms of total protein from both strains and 5, 2.5, and 1.25 μg of protein from the strain carrying 2μm-TBP were run in parallel, demonstrating that 2μm-TBP elevates the levels of TBP approximately eightfold over levels produced by the vector control.

FIG. 3

TBP overexpression does not suppress UAF mutation and does not increase the frequency of PSW variants. Leu⁺ colonies were picked directly from transformation plates containing strain NOY703 (rrn9Δ N-PSW) transformed with either the vector control (NOY1001) or 2μm-TBP (NOY1000). Colonies were suspended in water, and aliquots of 10-fold serial dilutions were then spotted on YEP-galactose (Gal) and YEPD (Glu). A total of nine independent colonies of each strain were analyzed, and three representative colonies of each transformation are shown here. The top three rows contain the control vector (pRS425), and the bottom three contain the 2μm-TBP plasmid (pNOY419). Plates were incubated for 8 days at 30°C.

FIG. 4

Primer extension analysis of rRNA precursors in UAF mutant strains overexpressing TBP. RNA was isolated from a wild-type (WT) strain (NOY388) and from rrn9Δ PSW strains (NOY902) carrying the vector control (pRS415), a CEN-TBP plasmid (pNOY347), or a 2μm (2μ)-TBP plasmid (pNOY419). Primer extension was then carried out on 3 μg of total RNA from the wild-type strain or 7 μg of total RNA from the other strains by using a ³²P-labeled rDNA primer, as described previously (20). The Pol II-derived rRNA precursors (with multiple start sites from upstream of the Pol I start site, +1; see reference 20) are present in all three rrn9Δ strains, and a Pol I-specific start site (+1) is not detectable.

FIG. 5

Decreased frequency of polymerase switching in rrn9Δ/rrn9Δ diploids and effects of TBP overexpression. (A) Aliquots of 10-fold serial dilutions of an rrn9Δ N-PSW strain (NOY703) and an rrn9Δ/rrn9Δ diploid strain (NOY1002) were spotted on YEP-galactose (Gal) and YEPD (Glu). The plates were incubated for 10 days at 30°C. (B) Primer extension analysis of rrn9Δ haploid and rrn9Δ/rrn9Δ diploid strains for measuring amounts of Pol II-derived rRNA transcripts. RNA was isolated from an rrn9Δ fob1Δ strain stably carrying ∼80 copies of rDNA (strain 1, NOY921) and from an rrn9Δ/rrn9Δ diploid strain (strain 2, NOY1002) grown on galactose medium (note that a fob1Δ derivative of the haploid rrn9Δ strain was used to stabilize ∼80 rDNA copy numbers because the fob1 deletion is known to prevent rDNA copy number expansion and contraction; see reference 17). Five micrograms of total RNA was subjected to primer extension analysis, as described previously (20). The rRNA transcripts derived from the GAL7-rDNA plasmid (GAL7) and from Pol II transcription of the chromosomal repeats (rDNA) are indicated. It should be noted that no Pol I-specific start site is detectable. The left panel (lanes 1 and 2) is from a 3-h exposure, and the right panel (lanes 1′ and 2′) is the same gel after a 16-h exposure. PhosphorImager quantitation of the Pol II-transcribed chromosomally derived bands showed an ∼3-fold decrease in the diploid strain relative to the haploid strain, per total amount of RNA (and per GAL7-35S rDNA-derived rRNA bands, which were approximately the same in both lanes). (C) TBP overexpression does not activate Pol I (or Pol II) transcription in rrn9Δ/rrn9Δ diploids. The rrn9Δ/rrn9Δ diploid strain (NOY1002) was transformed with the 2μm-TBP plasmid (pNOY419). Two independent transformants were then picked and aliquots of 10-fold serial dilutions were spotted on YEP-galactose and YEPD media. The plates were incubated for 11 days at 30°C.