Analysis of the interaction of the novel RNA polymerase II (pol II) subunit hsRPB4 with its partner hsRPB7 and with pol II - PubMed (original) (raw)

Analysis of the interaction of the novel RNA polymerase II (pol II) subunit hsRPB4 with its partner hsRPB7 and with pol II

V Khazak et al. Mol Cell Biol. 1998 Apr.

Abstract

Under conditions of environmental stress, prokaryotes and lower eukaryotes such as the yeast Saccharomyces cerevisiae selectively utilize particular subunits of RNA polymerase II (pol II) to alter transcription to patterns favoring survival. In S. cerevisiae, a complex of two such subunits, RPB4 and RPB7, preferentially associates with pol II during stationary phase; of these two subunits, RPB4 is specifically required for survival under nonoptimal growth conditions. Previously, we have shown that RPB7 possesses an evolutionarily conserved human homolog, hsRPB7, which was capable of partially interacting with RPB4 and the yeast transcriptional apparatus. Using this as a probe in a two-hybrid screen, we have now established that hsRPB4 is also conserved in higher eukaryotes. In contrast to hsRPB7, hsRPB4 has diverged so that it no longer interacts with yeast RPB7, although it partially complements rpb4- phenotypes in yeast. However, hsRPB4 associates strongly and specifically with hsRPB7 when expressed in yeast or in mammalian cells and copurifies with intact pol II. hsRPB4 expression in humans parallels that of hsRPB7, supporting the idea that the two proteins may possess associated functions. Structure-function studies of hsRPB4-hsRPB7 are used to establish the interaction interface between the two proteins. This identification completes the set of human homologs for RNA pol II subunits defined in yeast and should provide the basis for subsequent structural and functional characterization of the pol II holoenzyme.

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Figures

FIG. 1

FIG. 1

Structure of the hsRPB4 protein and gene. (A) Comparison of hsRPB4 predicted protein sequence with yeast RPB4 and putative mouse RPB4 subunits. Identical amino acid residues are shown in consensus. Conserved hydrophobic amino acids are indicated with ψ. (B) Genomic structure of hsRPB4. The full-length assembled cDNA for hsRPB4 is 1,902 bp. The genomic structure consists of four exons, which span ∼12 kb. Exons are represented as boxes, with translated sequence filled in black; introns are represented as lines. Underlined numbers represent the size of introns. Italicized numbers above the sequence represent amino acid positions relative to exon endpoints. Numbers below the sequence represent nucleotides of the hsRPB4 cDNA relative to exon-intron boundaries: in this case, 1 is taken to represent the first nucleotide of the longest cDNA obtained. The ORF encoding the hsRPB4 protein commences at 23 bp on this scale and extends 426 bp to position 449, followed by 1,453 bp of 3′ untranslated sequences. An Alu J family repeat is present at positions 919 to 1199 bp. The DNA sequence between 1498 and 1822 bp (shown hatched) is strongly homologous to the 40S ribosomal protein S26 and appears to be a novel S26 human pseudogene. An in-frame stop codon is present 65 bp upstream of position 1 of the cDNA, with no intervening splice sequences. The approximate endpoints of the three genomic clones used to generate sequence are shown above the diagram.

FIG. 2

FIG. 2

hsRPB4 RNA expression. A multitissue RNA blot was probed with either random primed hsRPB4 coding sequence (shown) or oligonucleotides specific to hsRPB4, with similar results. Reprobe of the same blot with an actin probe (40) confirmed equal load of all lanes.

FIG. 3

FIG. 3

hsRPB4 partial complementation of rpb4 null yeast. WY-4 containing pYES2 vector, pYES2-RPB4, or pYES2-hsRPB4 were diluted in suspension, and identical inocula dotted to plates were maintained for 2 days at 23, 30, 34, or 37°C as indicated.

FIG. 4

FIG. 4

Nuclear colocalization of hsRPB7 and hsRPB4. (A) Human HeLA cells which were starved (incubation in DMEM media with 0.5% fetal bovine serum for 72 h) (S) or exponentially growing (DMEM media with 10% of fetal bovine serum) (E), were fractionated as described in Materials and Methods. Total protein (2.5 μg) in the fractions indicated was resolved on an SDS–12% PAGE gel, and proteins were visualized by with rabbit polyclonal antibody against hsRPB7 followed by goat anti-rabbit antibody conjugated to horseradish peroxidase with enhanced chemiluminescence. The relative mobilities of the molecular size standards are indicated in kilodaltons. (B and C) Whole-cell lysates from Cos-1 cells transiently transfected with a pCMV vector (lane 1) or with pCMV vector expressing HA-tagged hsRPB4 (lane 2), native hsRPB7 (lane 3), HA-tagged hsRPB7 (lane 4), or both pCMV-hsRPB7 and pCMV-HA-hsRPB4 (lane 5) were resolved on duplicate SDS–12% PAGE gels. Blots were probed with rabbit anti-hsRPB7 polyclonal antibody (B) or rabbit polyclonal antibody against the HA tag (C). Proteins were visualized with goat anti-rabbit antibody conjugated to horseradish peroxidase as described in Materials and Methods. The estimated size for hsRPB7 is ∼27 kDa, that for HA-hsRPB7 is ∼32 kDa, and that for HA-hsRPB4 is ∼22 kDa. The mobilities of the molecular size standards are indicated in kilodaltons. (D) Cos-1 cells, transiently transfected with pCMV vector expressing a Myc-hsRPB4 fusion, were incubated 24 h in standard media and stained with DAPI (left) to visualize nuclei and with mouse monoclonal anti-Myc antibody followed by goat anti-mouse immunoglobulin G-rhodamine conjugate. Note that in approximately 10 to 20% of transfected cells HA-hsRPB4 localized to large granular structures in the nucleus. At this time, the most likely explanation for these structures is that they represent aggregates of overexpressed protein, although the possibility that they indicate the association of hsRPB4 with particular nuclear compartments, as reported for other pol II subunits (12, 65), has not been rigorously excluded.

FIG. 5

FIG. 5

Association of hsRPB4 and hsRPB7 in mammalian cells. Cos-1 cells transiently transfected with pCMV vector alone or with pCMV vector containing HA-hsRPB4 were immunoprecipitated with rabbit polyclonal antibody specific for HA. Whole-cell lysates (lanes 1 and 2) or immunoprecipitates (lanes 3 and 4) from Cos-1 cells transfected with pCMV vector (lanes 1 and 3) or with pCMV vector expressing HA-hsRPB4 fusion protein (lanes 2 and 4) were resolved on an SDS–12% PAGE gel. Endogenous and coimmunoprecipitated hsRPB7 proteins were visualized with rabbit polyclonal antibody to hsRPB7 as described above. The species migrating at ∼32 kDa in the two lanes of immunoprecipitation is nonspecific.

FIG. 6

FIG. 6

hsRPB4 and hsRPB7 are constituents of anti-CTD immunoaffinity-purified human pol II holoenzyme. Parallel samples of purified human polymerase II complex isolated by immunoprecipitation with anti-CTD (as described in reference 44) were resolved by SDS-PAGE. Left, silver stain of molecular size markers (MWM) and pol II to confirm purification; right, sample was blotted to membrane, probed with antibody to hsRPB4, and then reprobed without intervening strip with antibody to hsRPB7, confirming the presence of these proteins in the complex.

FIG. 7

FIG. 7

hsRPB4 and hsRPB7 are constituents of chromatographically purified transcriptionally active human pol II. (A) Fractions of high-pressure liquid chromatography DEAE-5PW column purified RNA polymerase II (44) were resolved by gradient gel and silver stained. M, molecular size marker; I, input; F, flow- through. The numbers across the top indicate fraction numbers. To the right are shown the positions of marker pol II subunits and approximate migration of hsRPB4 and hsRPB7 (compare with banding pattern in Fig. 6). (B) Parallel fractions resolved by SDS-PAGE and Western blotted, probed with combined antibodies to hsRPB4 and hsRPB7, and visualized with alkaline-phosphatase-conjugated secondary antibody. (C) Assay of nonspecific transcription activity of fractions based on incorporation of UTP into acid-insoluble material in 20 min with φX174 DNA as template, as described in reference .

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