RPAP1, a Novel Human RNA Polymerase II-Associated Protein Affinity Purified with Recombinant Wild-Type and Mutated Polymerase Subunits

Abstract

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.RNA polymerase II (RNAPII) is the multisubunit enzyme that synthesizes all mRNA precursors in eukaryotes. RNAPII is highly conserved among species, and in humans, RNAPII consists of 12 subunits, named Rpb1 to Rpb12 (16, 88). The two largest subunits, Rpb1 (220 kDa) and Rpb2 (140 kDa), form the enzyme's catalytic center and are homologous to the ␤Ј and ␤ subunits of bacterial RNAP, respectively. Five subunits, Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12, are also found in RNAPI and RNAPIII. Rpb3 and Rpb11 are homologous to the ␣ 2 homodimer involved in bacterial RNAP assembly. Rpb9 was attributed a role in elongation through its action at DNA arrest sites (3). In Saccharomyces cerevisiae, Rpb4 and Rpb7 form a subcomplex that can dissociate from the enzyme upon changes in environmental conditions. Under active growth conditions, most yeast RNAPII molecules do not contain the Rpb4-Rpb7 dimer, which primarily associates during the stationary phase or following stress (11). Functional studies of human RNAPII have been limited due to the lack of appropriate systems for purifying variant forms of the human enzyme.The availability of crystal structures of both yeast (17,18,26,30) and bacterial (8,57,58,84,89) RNAPs has been invaluable for understanding many of the molecular features of the transcription reaction. For example, the structure of elongating RNAPII has revealed the positioning of the RNA-DNA duplex during the transcription reaction (30). The structures available support a model in which the DNA enters the enzyme through a channel formed by a pair of "jaws" before accessing a deep cleft, at the bottom of which is buried the Mg 2ϩ -ion-containing active site; the DNA then turns by about 90°along a wall where the upstream end exits the enzyme (30). Many loops and helices either directly contact or closely approach the RNA-DNA duplex, thus suggesting putative functions for these structural elements in the transcription...

Role of Ostm1 Cytosolic Complex with Kinesin 5B in Intracellular Dispersion and Trafficking

“…The 30-aa CT cytosolic tail of mouse Ostm1 cDNA (93.3% identity with the human sequence) was cloned into the mammalian expression vectors pMZI and AB-0411 (23,24) in such a way that the C-terminal domain of Ostm1 carries a tandem affinity purification (TAP) tag at its C terminus and N terminus, respectively (25). Individual EcR293 stable cell lines were produced following transfection (Effectene; Qiagen), and expression of TAP-tagged Ostm1 cells was induced for 24 h with 1 M Ponasterone A (Invitrogen) (26). The TAP-tagged complexes were then purified and concentrated as described previously (25).…”

Mutational Analysis of Human RNA Polymerase II Subunit 5 (RPB5): The Residues Critical for Interactions with TFIIF Subunit RAP30 and Hepatitis B Virus X Protein

“…TFIIF binds directly to RNAPII and also helps to recruit the enzyme to the promoter (14,(25)(26)(27)(28). TFIIF can induce further bending and wrapping of the promoter DNA against the mobile clamp of RNAPII during the formation of the preinitiation complex (11,(29)(30)(31)(32).…”

Mapping the Disease Protein Interactome: Toward a Molecular Medicine GPS to Accelerate Drug and Biomarker Discovery

“…For this purpose, we like combining the use of HEK293 cells, for which we and others have already generated significant, although far from complete, protein-protein interaction data, with cell lines specifically originating from the disease/tissue of interest. Mapping interaction networks generally identifies both upstream (i.e., regulators) and downstream (i.e., regulated proteins) effectors of the tagged proteins, as we have shown for components of the transcription machinery [3][4][5]8,15,16,18 and as others have shown for proteins specifically involved in disease conditions. 2,6,10,12,27,32,33 Consequently, given that a protein associated with a specific disease condition represents a putative biomarker for this particular disease, its interactors putatively represent additional biomarkers that can regulate the disease phenotype by acting as upstream or downstream effectors.…”

“…15 Accordingly, these proteins were named RNAPII-Associated Proteins (RPAPs, namely, RPAP1, 2, 3, and 4). 3,8,15,16 The 77-bait data set (2009) revealed that RPAP3 is part of an 11-subunit protein complex containing POLR2E (RPB5), a subunit shared by all three nuclear RNAPs, and a set of factors previously characterized as being involved in protein complex assembly (according to their Gene Ontology [GO] term). 3 These include the chaperone cofactors RPAP3 and PIH1D1; the classical prefoldins PFDN2 and PFDN6; and the prefoldin-like proteins UXT, PDRG1, and C19orf2/URI.…”

“…15 Accordingly, these proteins were named RNAPII-Associated Proteins (RPAPs, namely, RPAP1, 2, 3, and 4). 3,8,15,16 The 77-bait data set (2009) …”