Iwr1 Protein Is Important for Preinitiation Complex Formation by All Three Nuclear RNA Polymerases in Saccharomyces cerevisiae

Background Iwr1, a protein conserved throughout eukaryotes, was originally identified by its physical interaction with RNA polymerase (Pol) II. Principal Findings Here, we identify Iwr1 in a genetic screen designed to uncover proteins involved in Pol III transcription in S. cerevisiae. Iwr1 is important for Pol III transcription, because an iwr1 mutant strain shows reduced association of TBP and Pol III at Pol III promoters, a decreased rate of Pol III transcription, and lower steady-state levels of Pol III transcripts. Interestingly, an iwr1 mutant strain also displays reduced association of TBP to Pol I-transcribed genes and of both TBP and Pol II to Pol II-transcribed promoters. Despite this, rRNA and mRNA levels are virtually unaffected, suggesting a post-transcriptional mechanism compensating for the occupancy defect. Conclusions Thus, Iwr1 plays an important role in preinitiation complex formation by all three nuclear RNA polymerases.

genomic library (ROSE et al. 1987) was used to transform iwr1-2 (UMY2299), rpc160-101 (UMY2304), and rpb5-101 (UMY2309) mutants, and transformants that could lose the RIT1 plasmid were identified. YCp50 plasmids isolated from transformants lacking RIT1 were partially sequenced, and the DNA sequence was used for homology searches against the S. cerevisiae genome. To confirm that the mutations in UMY2299 and UMY2304 were genetically linked to the IWR1 and RPC160 loci, we integrated a URA3 marker at the corresponding wild-type locus in rit1Δ strains, generating UMY2448 and UMY2332. These strains were crossed to each mutant (UMY2299 and UMY2304) and tetrad analysis showed co-segregation of the Ura + and sectoring phenotypes.
Plasmid constructions: DNA manipulations, plasmid preparations, and bacterial transformations were performed according to standard protocols. The plasmid pRS316-IWR1 (p1251) was constructed by cloning a HindIII/XbaI IWR1 fragment isolated from a library plasmid complementing iwr1-2 mutant into the corresponding sites of pRS316.
Plasmid pRS316-RPC160 (p1615) was constructed by cloning an AhaII/XhoI fragment from an YCp50 library plasmid complementing the rpc160-101 mutant, into the ClaI/XhoI sites of pRS316. For further information regarding plasmids used in this paper please contact the corresponding author.
Immunofluorescence: To localize Iwr1, cells were grown in 5 ml YEPD at 30° to an OD 600 of 0.3, 670 µl formaldehyde (37%) was added and the cells were incubated for 40 min at RT. Cells were collected and washed once with 1 x PBS, pH 7.4. Immunofluorescence was done as described (BURKE et al. 2000). The primary antibody, mouse anti-HA (12CA5), was diluted 1:2000. The secondary antibody, goat anti-mouse linked to Cy3 (PA43002, Amersham Biosciences), was diluted 1:200. Cells were viewed in a Zeiss Axioskope 50 microscope using a 100x objective. Images were acquired using a Hamamatsu-digital camera (C4742-95).
Polysome profiles: Cells were grown in 200 ml at 30° in selective medium to an OD 600 0.4. Cycloheximide was added (100 µg/ml) 5 min before transferring the culture to an ice water bath for 15 min. Cells were collected at 4°, washed twice in ice-cold Breaking buffer (Bb; 20 mM Tris-HCl pH 7.4, 10 mM MgCl 2 , 100 mM KCl, 0.5 mM DTT, 100 µg/ml cycloheximide). The cells were resuspended in 1 volume of Bb, followed by addition of 1 volume of glass beads, and cells were disrupted by 6 x 20 sec on a vortex mixer, and the insoluble material was pelleted by centrifugation at 10,000 x g for 5 min at 4°. The Supernatant was transferred to a microfuge tube and subjected to a second centrifugation at 10,000 x g for 20 min at 4°. The Supernatant was applied to a 12 ml linear 10 to 45 % sucrose gradient prepared in Bb lacking cycloheximide and centrifuged for 2.5 hrs at 40,000 rpm in a SW41 rotor at 4°. The gradients were collected from the top, and A 254 absorbances were monitored with the ISCO detection system. rtPCR: Total RNA was isolated from exponentially growing wild-type and iwr1∆ strains by the acid phenol method. The RNA was purified and treated with DNase I on Qiagen RNeasy columns, and first-strand cDNA synthesis was performed with random hexamers and Superscript III reverse transcriptase (Invitrogen) on 1 µg of total RNA. The relative representation of specific loci in this material was assayed by quantitative PCR in real-time on an Applied Biosystems 7500 machine.
In vivo labeling of RNA: In labeling experiments, cells were grown to an OD 600 of 0.8 in 120 ml SC-uracil medium at 30° before 125 µCi of 3 H-Uridine was added (33 Ci/mmol, Amersham Biosciences). Samples (20 ml) were collected after 0, 5, 10, 20, and 40 min. Total RNA was prepared and separated on an 8 % polyacrylamide 8 M urea gel. The gel was stained with ethidium bromide, quantified, soaked in NAMP100 Amplifyer for 15 min (Amersham Biosciences), dried onto 3 MM Whatman paper, and exposed to film.
Signals were quantified using QuantityOne-4.2.3 software (BioRad), and the rate of synthesis for each RNA type was calculated. For the pulse-chase experiment, cells were grown to an OD 600 of 0.8 in 50 ml SC-uracil medium at 30° and pulse-labeled using 125 µCi of 3 H-Uridine (33 Ci/mmol Amersham Biosciences) for 45 min. Cells were collected and resuspended in 250 ml pre-warmed SC medium containing excess (2 mM) uracil to begin the chase. Samples (40 ml) were collected after 0, 1, 2, 3, and 4 hrs. RNA was prepared, separated, and quantified as described above.
Immunoprecipitation: Cells were grown in 50 ml YEPD at 30° to an OD 600 of 0. Chromatin immunoprecipitation: Yeast strains BY4741 and the isogenic strain containing an iwr1 null allele RN3812 were obtained from Invitrogen. Cells were cultured in YEPD medium to an OD 600 of 0.6, crosslinked with 1 % formaldehyde, washed, and harvested as previously described (KURAS and STRUHL 1999). Total chromatin was isolated from these cells essentially as described (KURAS and STRUHL 1999) and sonicated to an average DNA fragment size between 300 and 500 bp. Chromatin immunoprecipitation was performed using antibodies against Tfc4, Rpc34 (both antibodies kindly supplied by Steve Hahn), Bdp1 (kindly provided by Ian Willis), and Rpb1 (8WG16 antibody from Covance).
Immunoprecipitated DNA and total input control DNA were assayed by real-time quantitative PCR using the Applied Biosystems 7300 Real-time PCR System.
Immunoprecipitation efficiency was determined for each locus by dividing the yield of PCR product in the immunoprecipitation sample by the amount of product obtained from the input control. Relative occupancy values were determined by dividing the immunoprecipitation 7 efficiency at each locus by the immunoprecipitation efficiency at a negative control locus (either the middle of the POL1 ORF or an ORF-free region of chromosome V). The occupancy value of the negative control, 1.0, was subtracted from all values to yield a baseline of 0. All occupancy values were normalized to set the occupancy of each factor in the wild-type strain at tC(GCA)B locus equal to 100 units. All experiments were performed a minimum of three times.