Expression of catalytically inactive Rat1_D235A increases 35S pre-rRNA accumulation.

<p>Kinetics of pre-rRNA labeling in <i>P_MET::RAT1</i> strains carrying the empty plasmid (shown in orange) or the plasmid expressing Rat1_D235A (shown in purple) following growth in the presence of methionine for 8h to repress expression of chromosomal Rat1. (A) Incorporation into 35S pre-rRNA. A higher plateau for 35S was observed in the strain expressing Rat1_D235A. (B–D) Incorporation into 20S, 27SA and 27SB pre-rRNAs, respectively. Differences in labeling kinetics for 20S, 27SA and 27SB pre-rRNAs were not significant (p>0.05) as shown by a student’s T-test. The T-test determines the probability that two samples come from the same population, and was performed based on a two-tailed distribution.</p

Time courses of phenotypes in strains depleted for Rat1.

<p>(A) OD₆₀₀ of the cultures at the time points indicated. Non-depleted cells, growing in the absence of methionine, were maintained in exponential growth phase by frequent dilution with pre-warmed medium. Rat1-depleted cells, growing in the presence of methionine, were diluted at the same times and to the same extent as non-depleted cells. OD₆₀₀ values (cell density) for the non-depleted strain at the time points indicated are shown in orange. OD₆₀₀ values for the Rat1-depleted strain at the same time points are indicated in purple. Doubling time for the non-depleted strain was ∼100 min. (B) Relative abundances of 5.8S(L) (Rat1-independent) and 5.8S(S) (Rat1-dependent), with total 5.8S rRNA abundance set to 100% at each time point. Graphs show the averages of three independent experiments.</p

Parameters used for modeled curves shown in Figure 2.

<p>V: Velocity of transcription in nucleotides (nt) incorporated sec⁻¹.</p><p>P: Probability that pre-rRNA will undergo nascent transcript cleavage (NTC) rather than released transcript cleavage (RTC).</p><p>NTC window: The distance traveled (in nt) by the transcribing polymerase downstream of site A2 prior to the NTC event.</p><p>Equilibration time: Time required for [³H] tritium uptake and equilibration of the internal nucleotide pool, prior to linear incorporation of label into newly synthesized RNA.</p><p>Lifetimes are in seconds.</p

Pre-rRNA processing pathways in budding yeast.

<p>(A) Structure of the 35S pre-rRNA, showing the location of cleavage site A2. (B) Pre-rRNA processing pathways via nascent transcript cleavage (NTC) and released transcript cleavage (RTC). The points at which Rat1 functions as a 5′ exonuclease are indicated.</p

Plasmids used in this project.

<p>Plasmids used in this project.</p

Comparison of predicted labeling curves with different levels of cotranscriptional pre-rRNA cleavage.

<p>(A) Modeled incorporation into 35S and 20S pre-rRNA with high (70%) cotranscriptional cleavage. (B) Modeled incorporation into 35S and 20S pre-rRNA with low (30%) cotranscriptional cleavage. Purple lines represent the NTC population. Orange lines represent the RTC population. The black line is the sum of the NTC and RTC populations.</p

Depletion of Rat1 inhibits NTC.

<p>Kinetics of pre-rRNA labeling in <i>P_MET3::RAT1</i> strains carrying the plasmid expressing Rat1 (shown in purple) or the empty plasmid (shown in orange), following growth in the presence of methionine for 8 h to repress expression of chromosomal Rat1. (A) Incorporation into 35S pre-rRNA. (B) Incorporation into 20S pre-rRNA. (C) Incorporation into 27SA pre-rRNA. (D) Incorporation into 27SB pre-rRNA. The three points shown for each time point represent the values obtained from three independent experiments. Solid lines represent the modeled response using the values from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085703#pone-0085703-t003" target="_blank">Table 3</a>. Values for Rat1-expression are shown in purple and values for Rat1-depletion in orange.</p

The SWI/SNF Chromatin Remodeling Complex Influences Transcription by RNA Polymerase I in <em>Saccharomyces cerevisiae</em>

<div><p>SWI/SNF is a chromatin remodeling complex that affects transcription initiation and elongation by RNA polymerase II. Here we report that SWI/SNF also plays a role in transcription by RNA polymerase I (Pol I) in <i>Saccharomyces cerevisiae</i>. Deletion of the genes encoding the Snf6p or Snf5p subunits of SWI/SNF was lethal in combination with mutations that impair Pol I transcription initiation and elongation. SWI/SNF physically associated with ribosomal DNA (rDNA) within the coding region, with an apparent peak near the 5′ end of the gene. In <i>snf6</i>Δ cells there was a ∼2.5-fold reduction in rRNA synthesis rate compared to WT, but there was no change in average polymerase occupancy per gene, the number of rDNA gene repeats, or the percentage of transcriptionally active rDNA genes. However, both ChIP and EM analyses showed a small but reproducible increase in Pol I density in a region near the 5′ end of the gene. Based on these data, we conclude that SWI/SNF plays a positive role in Pol I transcription, potentially by modifying chromatin structure in the rDNA repeats. Our findings demonstrate that SWI/SNF influences the most robust transcription machinery in proliferating cells.</p> </div

Strains used in this study.

<p>Strains used in this study.</p

A small accumulation of Pol I complexes in the 5′ end of rDNA in <i>snf6</i>Δ.

<p>(A) Distribution frequency for the number of polymerases per gene was revealed by EM analysis of Miller chromatin spreads in <i>snf6</i>Δ (DAS647) and WT (DAS648). (B) More than 100 rDNA genes from Miller chromatin spreads were analyzed in WT and <i>snf6</i>Δ. Pol I density and percentage of actively transcribed genes in <i>snf6</i>Δ and WT are similar. (C) Polymerase occupancy as a function of position within the transcribed region of rDNA in <i>snf6</i>Δ and WT cells. A small peak of Pol I occupancy in the 5′ end of rDNA in <i>snf6</i>Δ is indicated by an asterisk. All mappable genes in the dataset were analyzed, corresponding to >60 genes per strain and >2500 polymerases per strain. Schematic below the X-axis represents the Pol I transcribed region of the rDNA. (D) Same as C but an additional two WT control strains (NOY388 and BY4741) are plotted. BY4741 data are from El Hage et al. 2010 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056793#pone.0056793-ElHage1" target="_blank">[34]</a>.</p