Phosphorylation of the RNA polymerase II carboxy-terminal domain by the Bur1 cyclin-dependent kinase - PubMed (original) (raw)
Phosphorylation of the RNA polymerase II carboxy-terminal domain by the Bur1 cyclin-dependent kinase
S Murray et al. Mol Cell Biol. 2001 Jul.
Abstract
BUR1, which was previously identified by a selection for mutations that have general effects on transcription in Saccharomyces cerevisiae, encodes a cyclin-dependent kinase that is essential for viability, but none of its substrates have been identified to date. Using an unbiased biochemical approach, we have identified the carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II, as a Bur1 substrate. Phosphorylation of Rpb1 by Bur1 is likely to be physiologically relevant, since bur1 mutations interact genetically with rpb1 CTD truncations and with mutations in other genes involved in CTD function. Several genetic interactions are presented, implying a role for Bur1 during transcriptional elongation. These results identify Bur1 as a fourth S. cerevisiae CTD kinase and provide striking functional similarities between Bur1 and metazoan P-TEFb.
Figures
FIG. 1
The largest subunit of RNA Pol II (Rpb1) coimmunoprecipitates with Bur1. (A) Extracts were prepared from yeast cells expressing untagged Bur1 (lane 1), FLAG-Bur1 (lane 2), or FLAG–Bur1-3 (lane 3). FLAG-Bur1 and FLAG–Bur1-3 were immunoprecipitated, and the beads were washed using a Tris-NaCl buffer containing 450 mM NaCl. Immunoprecipitates were resolved by SDS-PAGE on a 7.5% acrylamide gel. Epitope-tagged Bur1 was detected using the M2 anti-FLAG antibody. (B) Immunoprecipitates from panel A were assayed for in vitro kinase activity by incubation with [γ-32P]ATP. The reaction products were resolved by SDS-PAGE on a 7.5% acrylamide gel. Arrows indicate the major Bur1-dependent phosphorylated products. (C) Western analysis of immunoprecipitates from panel A using the anti-Rpb1 E2 antibody, which was raised against the second exon of Drosophila Rpb1. In all panels, the presence or absence of the epitope tag is denoted by a “+” or “−,” respectively. Molecular weight markers are indicated on the right.
FIG. 2
Bur1 phosphorylates the Rpb1 CTD in vitro. (A) Bur1+ or FLAG-Bur1 was expressed in strains containing either Rpb1+ or the Rpb1Δ103 CTD truncation as indicated at the top. FLAG-Bur1 was immunoprecipitated using a Tris-acetate buffer, the immunoprecipitates were assayed for kinase activity by the addition of [γ-32P]ATP, and the products were separated in an SDS-PAGE (7.5% acrylamide) gel. The positions of Rpb1 and Rpb1Δ103 are indicated. (B) FLAG antibodies were used to immunoprecipitate Bur1 (lanes 1 and 4), FLAG-Bur1 (lanes 2 and 5), or FLAG–Bur1-3 (lanes 3 and 6) from cellular lysates. Immunoprecipitates were incubated with purified recombinant β-Gal (lanes 1 through 3) or β-Gal–CTD, and reactions were initiated by the addition of [γ-32P]ATP. Reaction products were resolved by SDS-PAGE in a 7.5% acrylamide gel, and phosphorylated products were visualized by autoradiography. The arrow indicates the position of phosphorylated β-Gal–CTD. Phosphorylation of β-Gal–CTD by Bur1 results in a smear of phosphorylated products reminiscent of the hyperphosphorylated forms of Rpb1.
FIG. 3
Serine specificity of Bur1 interactions. FLAG-Bur1 or FLAG–Bur1-3 was expressed in a BUR+ yeast strain (GY458) from pRU8 and pRU9 and immunoprecipitated (IP) using anti-FLAG beads. The immunoprecipitated material was then incubated in a kinase assay containing nonradioactive ATP. Samples were probed in Western blots using antibodies 8WG16 (specific for unphosphorylated CTD repeats), H14 (phosphoserine 5 specific), or H5 (phosphoserine 2 specific). Lanes 1 and 2 contain 100 μg of crude extract, while lanes 3 through 6 contain material immunoprecipitated from 1 mg of extract.
FIG. 4
RPB1-BUR1 genetic interactions. (A) Yeast strains with the indicated genotypes were streaked onto YPD media and grown at 30°C for 3 days. The bur1-2 rpb1_Δ_103 double mutants grew extremely poorly relative to the single mutants. (B) Yeast strains containing RPB1 or rpb1 CTD truncation alleles were transformed with either a 2μm vector (pRS426), a 2μm BUR1 plasmid (pGP112), or a 2μm bur1-3 plasmid (pGP211). Transformants were replica plated to an SC complete plate (left) and an SC plate lacking lysine (right) and were grown at 30°C for 3 days. The CTD truncations suppressed the bur1-3 Spt− phenotype. (C) Yeast strains with the indicated genotypes were replica plated to complete plates at 30 and 38.5°C and to complete plates lacking inositol.
FIG. 5
Genetic evidence for a Bur1 role during elongation. (A) 6AU sensitivity of CTD kinase mutants. Strains containing mutations shown on the left were replica plated to SC-Ura and SC-Ura plus 6AU plates. The photographs were taken after 2 days of growth at 30°C. (B) Temperature sensitivity of bur1-2 ppr2Δ double mutants. Strains GHY296 and FY886 were crossed, and representative progeny with the indicated genotypes, derived from a single tetrad, were streaked onto YPD plates and grown at 30 or 37°C for 2 days.
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