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RNA polymerase II subunit composition, stoichiometry, and phosphorylation. 总被引:25,自引:7,他引:18
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RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme. 相似文献
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Human RNA polymerase II subunit hsRPB7 functions in yeast and influences stress survival and cell morphology. 总被引:12,自引:0,他引:12
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V Khazak P P Sadhale N A Woychik R Brent E A Golemis 《Molecular biology of the cell》1995,6(7):759-775
Using a screen to identify human genes that promote pseudohyphal conversion in Saccharomyces cerevisiae, we obtained a cDNA encoding hsRPB7, a human homologue of the seventh largest subunit of yeast RNA polymerase II (RPB7). Overexpression of yeast RPB7 in a comparable strain background caused more pronounced cell elongation than overexpression of hsRPB7. hsRPB7 sequence and function are strongly conserved with its yeast counterpart because its expression can rescue deletion of the essential RPB7 gene at moderate temperatures. Further, immuno-precipitation of RNA polymerase II from yeast cells containing hsRPB7 revealed that the hsRPB7 assembles the complete set of 11 other yeast subunits. However, at temperature extremes and during maintenance at stationary phase, hsRPB7-containing yeast cells lose viability rapidly, stress-sensitive phenotypes reminiscent of those associated with deletion of the RPB4 subunit with which RPB7 normally complexes. Two-hybrid analysis revealed that although hsRPB7 and RPB4 interact, the association is of lower affinity than the RPB4-RPB7 interaction, providing a probable mechanism for the failure of hsRPB7 to fully function in yeast cells at high and low temperatures. Finally, surprisingly, hsRPB7 RNA in human cells is expressed in a tissue-specific pattern that differs from that of the RNA polymerase II largest subunit, implying a potential regulatory role for hsRPB7. Taken together, these results suggest that some RPB7 functions may be analogous to those possessed by the stress-specific prokaryotic sigma factor rpoS. 相似文献
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Structural and functional homology between the RNAP(I) subunits A14/A43 and the archaeal RNAP subunits E/F
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In the archaeal RNA polymerase and the eukaryotic RNA polymerase II, two subunits (E/F and RPB4/RPB7, respectively) form a heterodimer that reversibly associates with the core of the enzyme. Recently it has emerged that this heterodimer also has a counterpart in the other eukaryotic RNA polymerases: in particular two subunits of RNA polymerase I (A14 and A43) display genetic and biochemical characteristics that are similar to those of the RPB4 and RPB7 subunits, despite the fact that only A43 shows some sequence homology to RPB7. We demonstrate that the sequence of A14 strongly suggests the presence of a HRDC domain, a motif that is found at the C-terminus of a number of helicases and RNases. The same motif is also seen in the structure of the F subunit, suggesting a structural link between A14 and the RPB4/C17/subunit F family, even in the absence of direct sequence homology. We show that it is possible to co-express and co-purify large amounts of the recombinant A14/A43 heterodimer, indicating a tight and specific interaction between the two subunits. To shed light on the function of the heterodimer, we performed gel mobility shift assays and showed that the A14/A43 heterodimer binds single-stranded RNA in a similar way to the archaeal E/F complex. 相似文献
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Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly. 总被引:15,自引:9,他引:6
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Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II. 相似文献
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