Rpb3, Stoichiometry and Sequence Determinants of the Assembly into Yeast RNA Polymerase II in Vivo

Abstract

Stoichiometry of the third largest subunit (Rpb3) of the yeast RNA polymerase II is a subject of continuing controversy. In this work we utilized immunoaffinity and nickel-chelate chromatographic techniques to isolate the RNA polymerase II species assembled in vivo in the presence of the His 6 -tagged and untagged Rpb3. The distribution pattern of tagged and untagged subunits among the RNA polymerase II molecules is consistent with a stoichiometry of 1 Rpb3 polypeptide per molecule of RNA polymerase. Deletion of either ␣-homology region (amino acids 29 -55 or 226 -267) from the Rpb3 sequence abolished its ability to assemble into RNA polymerase II in vivo.Yeast Saccharomyces cerevisiae RNA polymerase II is a multisubunit enzyme comprised of 12 core polypeptides (1). With minor variations its subunit composition is characteristic of all eucaryal nuclear RNA polymerases and their archaeal counterparts (1, 2). Despite the trivial quantitative differences, a profound similarity can also be found between these eucaryal/ archaeal multisubunit enzymes and eubacterial RNA polymerases. The latter, being heterotetramers of the composition (␣) 2 ␤␤Ј, resemble eucaryal RNA polymerases in their overall appearance (3-6) and in the structural core composition, with the two largest procaryal subunits, ␤ and ␤Ј, having fairly conserved eucaryal orthologs, represented by yeast Rpb1 and Rpb2 (reviewed in Ref. 7). Less obvious homology was noted between eubacterial ␣ subunit and yeast Rpb3 (8), consistent with the reported functional equivalence of ␣ 2 and (Rpb3) 2 dimers in assembly of their respective enzymes (9).Young and co-authors also concluded, based on the [S 35 ]Met labeling of the RNA polymerase II subunits, that two copies of the subunits Rpb3, Rpb5, and Rpb9 were present in each RNA polymerase II molecule, whereas the rest were represented by a single polypeptide each or else recovered in submolar amounts (10). These inferred orthological relations between ␣ 2 and (Rpb3) 2 dimers were later questioned by the reports from several laboratories, that failed to detect homodimerization potential in yeast Rpb3 and its higher eucaryal homologs (11-13). Instead, in an array of in vitro assays, Rpb3 was shown to associate with another apparent ␣-subunit homolog, Rpb11, with a stoichiometry of 1:1 (11, 13). Human homologs of Rpb3 and 11 were also shown to associate in a complex of unknown stoichiometry in the yeast two-hybrid system (12). It was consequently suggested that the Rpb3-Rpb11 heterodimer serves as eucaryal analog of the ␣ 2 -homodimer (11); consistent with this hypothesis is the recovery of a Rpb2-Rpb3-Rpb11 core subassembly from a partially denatured Schizosaccharomyces pombe RNA polymerase II (14).In this work we utilized an independent method to ascertain the stoichiometry of the yeast Rpb3, based on simultaneous expression of the wild-type (genomic) RPB3 gene and its plasmid-borne His 6 -tagged version followed by affinity purification of the in vivo assembled RNA polymerase II species. EXPERIMENTAL PROCEDURESP...

Topology of Yeast RNA Polymerase II Subunits in Transcription Elongation Complexes Studied by Photoaffinity Cross-Linking

“…DNA templates with N 3 RdU positioned at the upstream edge of the footprinted region on the DNA, at -29/-28N, crosslinked to RPB3 and RPB7 ( Figure 6A, lane 2). The cross- linking to RPB3 seemed reasonable in light of RPB3 being structurally analogous to E. coli RNAP subunit R [phylogenetic tree and review by Svetlov et al (21); 43, [105][106][107][108]. The prokaryotic subunit interacts with DNA in the upstream region of some promoters (109)(110)(111)(112)(113), and the finding that RPB3 cross-linked from the upstream edge of the RNAP was consistent with the analogous functions of these eukaryotic and prokaryotic subunits.…”

Immunoaffinity Purification and Functional Characterization of Human Transcription Factor IIH and RNA Polymerase II from Clonal Cell Lines That Conditionally Express Epitope-tagged Subunits of the Multiprotein Complexes

“…In humans, RPB3 and RPB5 can each homodimerize and interact with almost all of the other subunits in vitro, suggesting that RPB3 and RPB5 may play a role in the assembly of pol II (12). However, the isolation of an RPB3-deficient yeast pol II indicates that RPB3, which shares sequence similarity with the ␣ subunit of prokaryotic RNA polymerase, is dispensable for complex assembly in vivo (17). The finding that human RPB5 can functionally interact with the pX protein of the hepatitis B virus and with TFIIB also suggests that RPB5 may act as a bridging factor between pol II and regulatory proteins (18,19).…”

Interaction of Fcp1 Phosphatase with Elongating RNA Polymerase II Holoenzyme, Enzymatic Mechanism of Action, and Genetic Interaction with Elongator

“…The antibodies to detect phosphorylated and nonphosphorylated RNAPII in crude chromatin were 8G3 (Pharmingen) and 8WG16 (Babco), respectively. The anti-Elp3, anti-Fcp1, and anti-Rpb3 antibodies have been described elsewhere (16,26,27).…”