Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Various complexes that contain the core subunits of RNA polymerase II associated with different transcription factors have been isolated from eukaryotes; their precise molecular constitution depends on the purification procedure. We estimated the numbers of various components of such complexes in an HeLa cell by quantitative immunoblotting. The cells were lysed with saponin in a physiological buffer; approximately 140,000 unengaged polymerases (mainly of form IIA) were released. Only approximately 4,000 of these soluble molecules sedimented in glycerol gradients as holoenzyme-sized complexes. About 180,000 molecules of polymerases (approximately 110,000 molecules of form IIO) and 10,000 to 30,000 molecules of each of TFIIB, TFIIEalpha, TFIIEbeta, TFIIF-RAP74, TFIIF-RAP30, and TFIIH-MAT1 remained tightly associated with the nuclear substructure. Most proteins and run-on activity were retained when approximately 50% of the chromatin was detached with a nuclease, but approximately 45,000 molecules of bound TATA binding protein (TBP) were detached. Similar results were obtained after cross-linking living cells with formaldehyde. The results provide little support for the existence of a large pool of soluble holoenzyme; they are consistent with TBP-promoter complexes in nuclease-sensitive chromatin being assembled into preinitiation complexes attached to the underlying structure.
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PMID:Quantitation of RNA polymerase II and its transcription factors in an HeLa cell: little soluble holoenzyme but significant amounts of polymerases attached to the nuclear substructure. 1040 29

The potent transactivator Tat recognizes the transactivation response RNA element (TAR) of human immunodeficiency virus type 1 and stimulates the processivity of elongation of RNA polymerase (Pol) II complexes. The cellular proteins Tat-SF1 and human SPT5 (hSPT5) are required for Tat activation as shown by immunodepletion with specific sera and complementation with recombinant proteins. In nuclear extracts, small fractions of both hSPT5 and Pol II are associated with Tat-SF1 protein. Surprisingly, the RAP30 protein of the heterodimeric transcription TFIIF factor is associated with Tat-SF1, while the RAP74 subunit of TFIIF is not coimmunoprecipitated with Tat-SF1. Overexpression of Tat-SF1 and hSPT5 specifically stimulates the transcriptional activity of Tat in vivo. These results suggest that Tat-SF1 and hSPT5 are indispensable cellular factors supporting Tat-specific transcription activation and that they may interact with RAP30 in controlling elongation.
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PMID:Tat-SF1 protein associates with RAP30 and human SPT5 proteins. 1045 43

Human transcription factor IIF (TFIIF) is an alpha(2)beta(2) heterotetramer of RNA polymerase II-associating 74 (RAP74) and RAP30 subunits. Mutagenic analysis shows that the N-terminal region of RAP74 between L155 (leucine at codon 155) and M177 is important for initiation. Mutants in this region have reduced activity in transcription, but none are inactive. Single amino acid substitutions at hydrophobic residues L155, W164, I176, and M177 have similar activity to RAP74(1-158), from which all but three amino acids of this region are deleted. Residual activity can be explained because each of these mutants forms a complex with RAP30 and recruits RNA polymerase II into the preinitiation complex. Mutants are defective for formation of the first phosphodiester bond from the adenovirus major late promoter but do not appear to have an additional significant defect in promoter escape. Negative DNA supercoiling partially compensates for the defects of TFIIF mutants in initiation, indicating that TFIIF may help to untwist the DNA helix for initiation.
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PMID:A region within the RAP74 subunit of human transcription factor IIF is critical for initiation but dispensable for complex assembly. 1052 26

RAP30, an RNA polymerase-associated protein (RAP) of approximately 30 kDa, is a component of the eukaryotic general transcription factor IIF (TFIIF). We have isolated a monoclonal antibody (MAb) that can be used to purify RAP30 under nondenaturing conditions. This MAb (designated 1RAP1) is a unique type of MAb that we have designated "polyol-responsive MAb." Polyol-responsive MAbs are high-affinity antibodies that release antigen in a buffer containing a low-molecular-weight polyhydroxylated compound (polyol) and a nonchaotropic salt. RAP30, contained on pET11d, was expressed in Escherichia coli by culturing and inducing protein expression at 26 degrees C. Under these conditions, approximately 50% of the RAP30 remains soluble. Inclusion bodies were removed from the cell lysate by centrifugation, the supernatant was treated with polyethyleneimine at 0.5 M NaCl to remove nucleic acids, and the soluble protein was applied directly to MAb-conjugated Sepharose. After extensive washing, RAP30 was eluted with buffer containing 0. 75 M ammonium sulfate and 40% propylene glycol. RAP30 produced by this procedure stimulates transcription from a minimal promoter. This is a rapid method for purifying unmodified RAP30 without renaturing the protein from inclusion bodies.
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PMID:Immunoaffinity purification of the RAP30 subunit of human transcription factor IIF. 1054 74

Transcription from the HIV-1 long terminal repeat (LTR) is regulated by the viral transactivator Tat, which increases RNA polymerase II (RNAP II) processivity. Previous reports have demonstrated that phosphorylation of the RNAP II carboxy-terminal domain by TFIIH and P-TEFb is important for Tat transactivation. Our present results demonstrate that phosphorylation of the RAP74 subunit of TFIIF is also an important step in Tat transactivation. Interestingly, while the general transcription factor TFIIF is required for both basal and Tat-activated transcription, phosphorylation of the RAP74 subunit occurs in the presence of Tat and correlates with a high level of transcription activity. Using a biotinylated DNA template transcription assay, we provide evidence that RAP74 is phosphorylated by TAF(II)250 during Tat-activated transcription. Depletion of RAP74 from the HeLa nuclear extract inhibited HIV-1 LTR-driven basal transcription and Tat transactivation. The addition of TFIIF, reconstituted from recombinant RAP30 and RAP74, to the depleted HeLa nuclear extract resulted in restoration of Tat transactivation. Of importance, the exogenous RAP74 was rapidly phosphorylated in the presence of Tat. These results suggest that RAP74 phosphorylation is one important step, of several, in the Tat transactivation cascade.
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PMID:Phosphorylation of the RAP74 subunit of TFIIF correlates with Tat-activated transcription of the HIV-1 long terminal repeat. 1070 53

General transcription factor IIF (TFIIF) is required for transcription by RNA polymerase II; it consists minimally of a heterodimer of RNA polymerase-associated proteins RAP30 and RAP74. According to solution and mutagenesis studies, the multiple domains of RAP30 and RAP74 bind PolII, TFIIB, TAF250 and DNA in interactions that are essential for transcription initiation and elongation. The X-ray structure of the RAP30/RAP74 interaction domains at 1.7 A resolution reveals a novel "triple barrel" dimerization fold and suggests with mutant data that interactions with the transcription apparatus are mediated not only by this tripartite beta-barrel, but also via flexible loops and alpha and beta-structures extending from it.
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PMID:Novel dimerization fold of RAP30/RAP74 in human TFIIF at 1.7 A resolution. 1118 78

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30 in vitro using purified recombinant proteins and in vivo in COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47-120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101-170) and the N-terminus (aa 1-100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding in in vitro and in vivo assays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.
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PMID:Direct interaction between the subunit RAP30 of transcription factor IIF (TFIIF) and RNA polymerase subunit 5, which contributes to the association between TFIIF and RNA polymerase II. 1127 33

RNA polymerase II-associating protein 74 (RAP74) is the large subunit of transcription factor IIF (TFIIF), which is essential for accurate initiation and stimulates elongation by RNA polymerase II. Mutations within or adjacent to the alpha1 helix of the RAP74 subunit have been shown to decrease both initiation and elongation stimulation activities without strongly affecting the interactions of RAP74 with the RAP30 subunit or the interaction between TFIIF and RNA polymerase II. In this manuscript, mutations within the alpha1 helix are compared with mutations made throughout the neighboring conserved N-terminal domain of RAP74. Changes within the N-terminal domain include disruptions of specific contacts with the alpha1 helix, which were revealed in the recently published x-ray crystal structure (Gaiser, F., Tan, S., and Richmond, T. J. (2000) J. Mol. Biol. 302, 1119-1127). Contacts between the beta4-beta5 loop and the alpha1 helix are shown to be largely unimportant for alpha1 helix function. Other mutations throughout the N-terminal domain are consistent with the establishment of the dimer interface with the RAP30 subunit. The RAP74-RAP30 interface is important for TFIIF function, but no particular RAP74 amino acids within this region have been identified that are required for TFIIF activities. The molecular target of the alpha1 helix remains unknown, but our studies refocus attention on this important functional motif of TFIIF.
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PMID:A key role for the alpha 1 helix of human RAP74 in the initiation and elongation of RNA chains. 1235 69

RMP was reported to regulate transcription via competing with HBx to bind the general transcription factor IIB (TFIIB) and interacting with RPB5 subunit of RNA polymerase II as a corepressor of transcription regulator. However, our present research uncovered that RMP also regulates the transcription through interaction with the general transcription factors IIF (TFIIF), which assemble in the preinitiation complex and function in both transcription initiation and elongation. With in vitro pull-down assay and Far-Western analysis, we demonstrated that RMP could bind with bacterially expressed recombinant RAP30 and RAP74 of TFIIF subunits. In the immunoprecipitation assay in COS1 cells cotransfected with FLAG-tagged RMP or its mutants, GST-fused RAP30 and RAP74 were co-immunoprecipitated with RMP in approximately equal molar ratio, which suggests that RAP30 and RAP74 interact with RMP as a TFIIF complex. Interestingly both RAP30 and RAP74 interact with the same domain (D5) of the C-terminal RMP of 118-amino-acid residuals which overlaps with its TFIIB-binding domain. Internal deletion of D5 region of RMP abolished its binding ability with both subunits of TFIIF, while D5 domain alone was sufficient to interact with TFIIF subunits. The result of luciferase assay showed that overexpression of RMP, but not the mutant RMP lacking D5 region, suppressed the transcription activated by Gal-VP16, suggesting that interaction with TFIIF is required for RMP to suppress the activated transcription. The interaction between RMP and TFIIF may be an additional passway for RMP to regulate the transcription, or alternatively TFIIF may cooperate with RPB5 and TFIIB for the corepressor function of RMP.
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PMID:Interaction with general transcription factor IIF (TFIIF) is required for the suppression of activated transcription by RPB5-mediating protein (RMP). 1273 19

The role of the RAP74 alpha1 helix of transcription factor IIF (TFIIF) in stimulating elongation by human RNA polymerase II (RNAP II) was examined using millisecond-phase transient-state kinetics. RAP74 deletion mutants RAP74(1-227), which includes an intact alpha1 helix, and RAP74(1-158), in which the alpha1 helix is deleted, were compared. Analysis of TFIIF RAP74-RAP30 complexes carrying the RAP74(1-158) deletion reveals the role of the alpha1 helix because this mutant has indistinguishable activity compared to TFIIF 74(W164A), which carries a critical point mutation in alpha1. We report adequate two-bond kinetic simulations for the reaction in the presence of TFIIF 74(1-227) + TFIIS and TFIIF 74(1-158) + TFIIS. TFIIF 74(1-158) is defective because it fails to promote forward translocation. Deletion of the RAP74 alpha1 helix results in increased occupancy of the backtracking, cleavage, and restart pathways at a stall position, indicating reverse translocation of the elongation complex. During elongation, TFIIF 74(1-158) fails to support detectable nucleoside triphosphate (NTP)-driven translocation from a stall position and is notably defective in supporting bond completion (NTP-driven translocation coupled to pyrophosphate release) during the processive transition between bonds.
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PMID:Human RNA polymerase II elongation in slow motion: role of the TFIIF RAP74 alpha1 helix in nucleoside triphosphate-driven translocation. 1583 64


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