Rtr1 interacts with the intact RNAPII complex. A. Summary of five different MudPIT analyses of the baits given at the top. The sequence coverage and total number of unique peptides for each subunit of interest are shown to the left. B. Western blot analysis of anion-exchange fractions obtained following separation of Rpb3-TAP complexes using antibodies directed against the unmodified CTD (UM CTD), S5-P CTD, Anti-CBP (Rpb3), or Anti-FLAG (Rtr1). C. Heat map representation of SAF values obtained from MudPIT analysis of each fraction (top, corresponding to Figure 1B). The highest SAF values are indicated in bright yellow and the absent values indicated in black, as shown in the intensity scale.

Rtr1 localizes to open reading frames. A. ChIP assays were performed using antibodies directed against different forms of RNAPII and Rtr1 as indicated. Quantitation of the S5-P (black circles) and S2-P (blue squares) occupancy compared to the occupancy of Rtr1 (red triangles) across the PMA1 genomic loci. B. Quantitation of protein occupancy across the PYK1 genomic loci as performed above. These data are shown as the average percent maximum IP ± standard deviation for comparison purposes between the different antibodies (n=3). The midpoint of the PCR amplicon was used as the distance from the ATG. A schematic representation of the genomic loci for each gene is shown below the graphs to illustrate the approximate location of the promoter and polyadenylation regions.

Deletion of Rtr1 causes transcription defects in vivo. A. The level of RNAPII occupancy decreases in rtr1Δ. Quantitation of the total RNAPII (measured by Rpb3) occupancy PMA1 genomic loci in both WT (black diamonds) and rtr1Δ (open diamonds) deletion strains. B. Quantitation of Rpb3 occupancy across the PYK1 genomic loci as performed above. Data is expressed as average fold over background ± standard deviations (n=3). C. Northern blot analysis of total RNA isolated from either WT or rtr1Δ cells as indicated. Northern blots (left panel) were visualized with probes directed against the RNAPII transcript PMA1 followed by probes directed against the RNAPIII transcript SCR. The 18S and 25S rRNA were visualized through ethidium bromide staining. D. Quantitation of three replicate northern blot analyses were performed by ImageQuant and are expressed as the average intensity of PMA1 normalized to the level of SCR1 or 18S rRNA ± standard deviations. E. Quantitative RT-PCR analysis from either WT or rtr1Δ total RNA for PMA1, PYK1, and ACT. Transcript levels are expressed as the average total ng of transcript normalized to the total ng of the RNAPIII transcript SCR1 ± standard deviations. F. Northern blot analysis of polyA mRNA isolated from either WT or rtr1Δ strains using probes directed against the 3’ end of NRD1 or the entire coding region of MRPL17. The location of the different transcripts is shown to the left of each panel and a schematic of the NRD1/MRPL17 loci is shown for visualization at the bottom.

Rtr1 can dephosphorylate RNAPII that is present in a ternary complex in vitro. A. Schematic representation of the ternary complex substrate assembled on a doubled stranded oligonucleotide with a 3’ overhang using Rpb3-TAP purified RNAPII. B. Rtr1 phosphatase reactions performed on 5 pmol of TFIIK ³²P-labeled ternary complex. The concentration of Rtr1 in each reaction is indicated at the top of the panel. C. Rtr1 phosphatase reactions performed on 5 pmol of MAPK2 ³²P-labeled ternary complex. The reactions in B and C were stopped by the addition of 6x SDS-loading buffer and were separated by SDS-PAGE. Bands were visualized by autoradiography. D. Quantitation of replicate phosphatase reactions as shown in B and C were performed using ImageQuant. Values are expressed as average intensity normalized to the input sample ± standard deviations.

Rtr1 associates with transcriptionally active RNAPII. A. In vitro transcription reactions were performed using increasing concentrations of Rpb3-TAP or Rtr1-TAP as shown. The full length (FL) RNA transcript is indicated to the right. The volume and intensity of the bands was measured using ImageQuant and the percent intensity compared to the maximum level is given at the bottom of the figure. B. Western blot analysis using monoclonal antibodies directed against the different forms of the RNAPII CTD as shown. Approximately 30µg of whole cell yeast extract (WCE) was analyzed for comparison to Rtr1-TAP purifications. C. Western blot analysis on CTD peptide binding assays visualized using antibodies against HA. The CTD peptides used were either unmodified (UM) or S5-P, S2-P, or S2,5-P. D. Quantitation of western blots represented in C. was performed by ImageQuant on four biological replicates and is expressed as the average binding intensity normalized to the UM CTD peptide ± standard deviation.

Rtr1 deletion causes increased RNAPII CTD S5-P in vivo. A. Western blot analysis of whole cell extracts of wild type (WT) compared to rtr1Δ. B. Quantitation of western blots represented in A. The data are shown as average fold enrichment over wild type ± standard deviations (n=3). Quantitation of the fold enrichment of S5-P RNAPII occupancy across the PMA1 (C) and PYK1 (D) open reading frames using the H14 monoclonal antibody. The midpoint of the PCR amplicon was used as the distance from the PMA1/PYK1 ATG. Data is expressed as average fold enrichment ± standard deviation (n=3), which was calculated by dividing the percent IP of each region over the percent IP of a control region on chromosome 5 where RNAPII does not bind.

Rtr1 is a CTD phosphatase. A. In vitro phosphatase reactions using 1 ug of TFIIK ³²P-labeled biotinylated CTD peptide (containing four YSPTSPS repeats) as substrate and increasing concentrations of purified recombinant Rtr1. The CTD peptide was resolved on a 16% Tricine gel which was then dried and exposed to a phosphorimaging screen. B. Western blot analysis of Rtr1 phosphatase reactions performed using CTDK-I labeled GST-CTD as a substrate. Reactions were visualized using antibodies directed against S5-P, S2-P, or HA. Approximately 5 pmol of GST-CTD was used as a substrate in each reaction using increasing concentrations of Rtr1. C. Western blot analysis of Rtr1 phosphatase reactions performed as in B, except that the GST-CTD substrate was modified by MAPK2 prior to the phosphatase experiments. *Quantitation for the MAPK2 experiments was performed on the slower migrating bands as illustrated. D. Quantitation of three replicate phosphatase reactions performed on either CTDK-I or MAPK2 modified GST-CTD. Quantitation was performed using ImageQuant and is expressed as the average percent maximum intensity ± standard deviations. E. Western blot analysis of whole cell extracts from wild-type and Rtr1 mutant strains. Cells were grown in SD Ura- media and total cellular protein was extracted with approximately 10, 50, and 100 µg of total protein loaded. The BY4741 and rtr1Δ strains were transformed with the URA3 containing plasmid pRS416. Proteins were visualized using antibodies directed against S5-P, Pgk1, Rpb3 (monoclonal) or Rtr1-TAP (TAP tag detected using Peroxidase anti-peroxidase). F. Quantitation of three replicate western blots as show in E. using ImageQuant software. Data is expressed as the average percent of wild type (BY4741) S5-P intensity normalized to the level of Pgk1 as a loading control ± standard deviation.