RNA polymerase switch in transcription of yeast rDNA: role of transcription factor UAF (upstream activation factor) in silencing rDNA transcription by RNA polymerase II

Abstract

Transcription factor UAF (upstream activation factor) is required for a high level of transcription, but not for basal transcription, of rDNA by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. RRN9 encodes one of the UAF subunits. We have found that rrn9 deletion mutants grow extremely slowly but give rise to faster growing variants that can grow without intact Pol I, synthesizing rRNA by using RNA polymerase II (Pol II). This change is reversible and does not involve a simple mutation. The two alternative states, one suitable for rDNA transcription by Pol I and the other favoring rDNA transcription by Pol II, are heritable not only in mitosis, but also in meiosis. Thus, S. cerevisiae has an inherent ability to transcribe rDNA by Pol II, but this transcription activity is silenced in normal cells, and UAF plays a key role in this silencing by stabilizing the first state.

Figure 1

RNA polymerase switch variants. (A) An unstable rrn9Δ haploid segregant (NOY684) derived from RRN9/rrn9Δ diploid strain NOY678. A heterogeneous cell population recovered from a tiny rrn9Δ colony formed on a tetrad-dissection plate was suspended in H₂O, spread on a YEPD plate, and incubated at 30°C for 7 days. (B) An isolated larger colony (left) and an isolated small colony (right) from the plate shown in A were restreaked on YEPD and incubated at 30°C for 7 days. (C) Non-PSW strain NOY703 and PSW strain NOY878 derived from NOY703 were streaked on YEP-galactose (Gal) and YEPD (Glu) plates and incubated at 30°C for 5 days.

Figure 2

Identification of 5′ ends of primary rRNA transcripts and evidence for transcription by Pol II in PSW strains. (A) RNA samples from control strain NOY505 (lane 1) and PSW strain NOY794 (lane 2) were analyzed by primer extension using a ³²P-labeled primer. The sequence ladder was obtained by dideoxy sequencing using the same primer and pNOY3237 DNA as template. Major start sites are indicated by ←. A dot marks the band in lane A corresponding to the published Pol I start site. (B) RNA samples were prepared from NOY878 (Δ9, PSW), NOY900 (Δ9, PSW, rpb1–1), NOY556 (WT), and NOY898 (rpb1–1) with and without temperature shift from 25° to 35° as indicated. Primer extension was carried out in duplicate as in A. Quantification of the sum of all Pol II-specific bands from −9 to −95 was done for NOY878 and NOY900 and that of the Pol I-specific band at +1 was done for NOY556 and NOY898 by using PhosphorImager analysis. A significant decrease (≈3-fold) of rRNA synthesis after temperature shift-up was observed for NOY900 but not for the other strains (see below). Gels shown are those visualized by a PhosphorImager. Four to seven reverse transcription analyses similar to that shown in the figure were done by using two independent RNA preparations for each strain. The values obtained for the ratio of rRNA synthesis at 35°C to that at 25°C are: NOY900, 0.33 ± 0.07; NOY878, 1.01 ± 0.29; NOY898, 0.80 ± 0.09; and NOY556, 0.83 ± 0.19.

Figure 3

(A) Loss of the PSW phenotype on reintroduction of the missing RRN9 into an rrn9Δ PSW strain. Plasmid pNOY446 carrying RRN9 (and URA3) or control URA3 plasmid pRS316 was introduced into NOY852 (rrn9Δ, PSW) by transformation. Four of the transformants isolated as Ura⁺ from each transformation were streaked on YEPD medium and then streaked on SD with and without 5-FOA (1 mg/ml). The plates were incubated at 30°C for 4 days. (B) A summary of experiments showing reversible switching between the non-PSW and PSW states.

Figure 4

Analysis of diploid clones from a cross between non-PSW and PSW strains. Strain NOY703 (non-PSW; N) and NOY877 (PSW; P) were crossed by plate-mating. Three diploid clones together with haploid parents (N and P) were analyzed for PSW phenotype by spotting aliquots of 10-fold serial dilutions of colonies on YEP-galactose (Gal) and YEPD (Glu), and the results are shown in A. Clones 1, 2, and 3 were sporulated, and tetrads were dissected on YEP-galactose. Colonies from tetrads showing four viable ones were analyzed for their PSW phenotype by spot test. The two original haploid parents (N and P) also were analyzed as controls. One tetrad from clone 1 (B), one tetrad from clone 3 (C), and two tetrads from clone 2 (D and E) are shown.