RNA Polymerase II Subunits 2, 3, and 11 Form a Core Subassembly with DNA Binding Activity

Abstract

RNA polymerase II purified from the fission yeast Schizosaccharomyces pombe consists of 10 species of subunit polypeptide. We introduced a histidine cluster tag sequence into the chromosomal rpb1 and rpb3 genes, which encode subunit 1 (Rpb1) and subunit 3 (Rpb3), respectively, and purified the RNA polymerase by Ni 2؉ affinity chromatography. After stepwise dissociation of the Rpb1-and Rpb3-tagged RNA polymerases fixed on Ni 2؉ -resin by increasing concentrations of urea or guanidium hydrochloride, Rpb2-Rpb3-Rpb11 or Rpb2-Rpb3-Rpb11-Rpb10 complexes were obtained. Since the complex consisting of Rpb2, Rpb3, and Rpb11 cannot be dissociated even after treatment with 6 M urea buffer, we propose that this complex represents a core subassembly of the RNA polymerase II, analogous to the ␣ 2 ␤ complex in the assembly of Escherichia coli RNA polymerase. Both the Rpb2-Rpb3-Rpb11 complex and the free Rpb1 protein showed DNA binding activity, although the affinity was weaker compared with the intact RNA polymerase.Eukaryotic nuclear RNA polymerases are all multisubunit enzymes consisting of some 10 species of polypeptide. Among three types of nuclear RNA polymerase, RNA polymerase II (pol II) 1 is involved in the synthesis of mRNA and thus plays a central role in gene transcription. The genes coding for all the putative subunits have been cloned and sequenced for three organisms, human, the budding yeast Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe; but for all these enzymes, little is known about the function(s) of each polypeptide.DNA blotting, ultraviolet cross-linking, and antibody inhibition studies indicated the involvement of subunit 1, the homologue of the E. coli RNA polymerase ␤Ј subunit, in DNA binding (1-5), whereas subunit 2, the homologue of the E. coli ␤ subunit, has binding sites for nucleotide substrates (6 -9) and DNA (1, 10, 11). S. cerevisiae pol II subunit 3 (RPB3) and the corresponding subunits from other organisms have a limited sequence similarity to the E. coli ␣ subunit. As in the case of the E. coli ␣ subunit, one enzyme protomer probably contains two molecules of RPB3, and this subunit forms a complex with RPB2 in an early step of the enzyme assembly in vivo (12, 13).The ␣-like sequence also exists in S. cerevisiae RPB11 and the corresponding subunits in other eukaryotes (14 -16). The RPB3 and RPB11 homologues of Arabidopsis thaliana pol II associate in vitro with each other (15). Molecular interaction between S. cerevisiae AC40 and AC19, which are RPB3 and RPB11 homologues present in both RNA polymerases I and III, was also suggested by genetic analysis (17).pol II purified from S. pombe contains 10 species of subunit (18), named Rpb1 to Rpb12 after the S. cerevisiae subunit nomenclature (19), lacking two subunits corresponding to RPB4 and RPB9 of S. cerevisiae pol II. The S. pombe genes coding for all 10 subunits have been cloned and sequenced (16, 20 -25). In the case of S. cerevisiae pol II, RPB4 and RPB7 are readily dissociated from the enzyme and form a heterodi...

Subunit D of RNA Polymerase from Methanosarcina acetivorans Contains Two Oxygen-labile [4Fe-4S] Clusters

“…One plausible role for the [4Fe-4S] clusters is to regulate the assembly of RNAP in response to cellular conditions, and this is supported by previous studies documenting archaeal subunit D, bacterial ␣ subunit, and eukaryotic Rpb3 as key subunits that initiate the assembly of RNAP (9,10,37,38). The D/␣/Rpb3 subunits are not directly involved in binding of template DNA and synthesis of RNA.…”

“…The [4Fe-4S] clusters in RNAP may serve a similar role. Based on previous structure-function studies of bacterial ␣ N-terminal domain (36,37), eukaryotic Rpb3 (32,38), and archaeal D of RNAPs (2) Fig. 10.…”

Mediator Influences Schizosaccharomyces pombe RNA Polymerase II-dependent Transcription in Vitro

“…The enzyme is composed of 12 subunits, Rpb1 to Rpb12, similar to what has been described for the budding yeast S. cerevisiae and mammalian cells (2,3). Biochemical analysis of the enzyme has revealed the presence of several distinct subcomplexes and has proved important in the analysis of the recently published x-ray crystallography structure of S. cerevisiae pol II (4,5). Previous characterization of S. pombe TFIIH has only concerned a specific subcomplex of this transcription factor, containing the Mcs6, Mcs2, and Pmh1 gene products (6).…”

Inter- and Intrasubunit Interactions during the Formation of RNA Polymerase Assembly Intermediate

“…coli RNAP assembles in vivo and in vitro according to the following scheme: ␣-␣ 2 -␣ 2 ␤-␣ 2 ␤␤Ј (9). The ␣ 2 ␤ assembly intermediate appears to be evolutionary conserved, and an ␣ 2 ␤-like RNAP II subassembly was isolated from yeasts (10,11). Further, mutations in E. coli ␣, and in its yeast RNAP II counterpart, Rpb3, that affect the ␣ 2 ␤ formation in their respective systems occur in homologous positions (12,13).…”