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

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