Characterization of cDNA for the large subunit of the transcription initiation factor TFIIF

T, Aso; Vasavada, HA; Kawaguchi, Tatsushi; Fj, Germino; Ganguly, Subinay; Kitajima, Shigetaka; Weissman, S; Yasukochi, Yukio

doi:10.1038/355461a0

Cited by 80 publications

(54 citation statements)

References 18 publications

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“…We then proceeded to carry out transcription in reactions containing purified, recombinant dTFIIB, dTBP, hTFIIE Peterson et al 1991;Sumimoto et al 1991), and hTFIIF (Sopta et al 1989;Aso et al 1992;Finkelstein et al 1992), along with purified RNA polymerase II from Drosophila embyros and the native (impure) fractions in the Drosophila transcription system (Wampler et al 1990). The purity of each of the recombinant proteins and the RNA polymerase II is shown in Figure 1A.…”

Section: Resultsmentioning

confidence: 99%

“…Depending on the reaction conditions, the requirement for TFIIA in basal transcription has been observed to be strong, weak, or nonexistent (Sawadogo and Roeder 1985;Wampler et al 1990;Cortes et al 1992;Ranish et al 1992;Sayre et al 1992). Notwithstanding, recent work has led to the purification and cloning of TFIIA, TFIIB, TBP (the TATA box-binding polypeptide of the native TFIID complex; Pugh and Tjian 1992), TFIIE, TFIIF, two subunits of TFIIH, and TFIIS [see references cited in Zawel andReinberg (1992, 1993) and Drapkin et al (1993) in addition to Aso et al 1992;Finkelstein et al 1992;Fischer et al 1992;Gileadi et al 1992].…”

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confidence: 99%

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Identification of a minimal set of proteins that is sufficient for accurate initiation of transcription by RNA polymerase II.

Tyree¹,

George²,

Lira-DeVito³

et al. 1993

Genes & Development

141

114

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In eukaryotes, initiation of mRNA synthesis is a multistep process that is carried out by RNA polymerase II and auxiliary factors that are commonly referred to as basal or general factors. In this study accurate initiation of transcription was reconstituted with purified, Escherichia coil-synthesized TFIIB, TBP (the TATA box-binding polypeptide of the TFIID complex), and the 30-kD subunit of TFIIF (also known as RAP30), along with purified, native RNA polymerase II from Drosophila embryos, calf thymus, or HeLa cells. This minimal set of factors was able to transcribe a subset of the promoters tested. The addition of both subunits of TFIIE and the 74-kD subunit of TFIIF increased the efficiency of transcription by a factor of 2 to 4. In contrast, the inclusion of a crude TFIID fraction from Drosophila embryos in place of recombinant TBP resulted in a strong dependence on TFIIE. By gel mobility-shift analysis, TFIIB, TBP, RAP30, and polymerase were able to assemble into DB and DBPolF30 complexes with transcriptionally competent (wild type or initiator mutant), but not with transcriptionally inactive (TATA and TATA/initiator mutant), versions of the Drosophila Adh promoter. Thus, it appears that RNA polymerase II is able to initiate transcription subsequent to assembly of the DBPolF30 complex, which is a minitranscription complex that represents the central core of the RNA polymerase II transcriptional machinery.[Key Words: RNA polymerase II; basal transcription factors; TFIIB; TATA box-binding protein; TFIIF (RAP30/74); in vitro transcription]

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Identification of a minimal set of proteins that is sufficient for accurate initiation of transcription by RNA polymerase II.

Tyree¹,

George²,

Lira-DeVito³

et al. 1993

Genes & Development

141

114

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“…RAP74 and RAP30 also have similar C-terminal regions with winged helix-turn-helix structures (2,3). The larger size of the human RAP74 subunit can be attributed to an extensive loop rich in Gly, Pro, Ser, Thr, and charged residues separating more structured N-and C-terminal domains (4,5).…”

Section: Tfiifmentioning

confidence: 99%

“…Human RAP74 homologues. A proposed alignment of TFIIF large subunits from human (Hs) (4,5), Drosophila (Dm) (22,23), Caenorhabditis elegans (Ce) (AAB00717), Arabidopsis thaliana (At) (NP192998), Schizosaccharomyces pombe (Sp) (T41039) and Saccharomyces cerevisiae (Sc) (24,25). Secondary structures from human RAP74 are indicated below the sequences.…”

Section: Radical Mutagenesis Of the N-terminal Domain Of Humanmentioning

confidence: 99%

A Key Role for the α1 Helix of Human RAP74 in the Initiation and Elongation of RNA Chains

Funk¹,

Nedialkov

et al. 2002

Journal of Biological Chemistry

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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 ␣1 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 ␣1 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 ␣1 helix, which were revealed in the recently published x-ray crystal structure (Gaiser, F., Tan TFIIF1 appears to be an ␣␤ heterodimer of RAP74 and RAP30 subunits, and previous reports that TFIIF may be an ␣ 2 ␤ 2 heterotetramer are not supported by the x-ray crystal structure (1). Although not evident from primary sequence, RAP74 and RAP30 subunits are structurally similar, with an intricate series of N-terminal␤-sheets that form a RAP74-RAP30 dimer interface. RAP74 and RAP30 also have similar C-terminal regions with winged helix-turn-helix structures (2, 3). The larger size of the human RAP74 subunit can be attributed to an extensive loop rich in Gly, Pro, Ser, Thr, and charged residues separating more structured N-and C-terminal domains (4, 5).TFIIF is an RNA polymerase II-specific transcription factor restricted to the eukaryotic kingdom. Fig. 1, therefore, shows an amino acid sequence alignment of the N-terminal regions of several RAP74 homologues spaced throughout eukaryotic evolution. Beneath the alignment, the primary sequence is correlated with regions of secondary structure. Regions of ␣-helix and ␤-sheet are derived from the crystal structure of human TFIIF (1) or else from secondary structure predictions (6 -9) in the regions where structural information is not available. The x-ray crystal structure indicates a unique dimer interface made up of RAP74 ␤-sheets ␤1, ␤2, ␤3, ␤6, ␤7, ␤8, and the corresponding ␤-sheets in RAP30. The ␤4 and ␤5 sheets of RAP74 interact to form the structured base of a loop that diverges from the dimer core. In the crystal structure, the RAP74 ␣1 helix makes intimate contacts with the ␤4 and ␤5 sheets.The ␣1 helix has been shown previously to be highly sensitive to mutation (10, 11). Several single amino acid changes, particularly in hydrophobic residues, cause significant defects in both accurate initiation and elongation. Because of symmetrical effects on initiation and elongation, ␣1 should function by contacting a molecular target common to both processes, which would implicate RNA polymerase II, TFIIF, and DNA as the most likely targets. Mutations in ␣1 do not appear to affect interaction with the RAP30 subunit, and no RAP30 contacts are identified for ␣1 in the crystal structure (1). As far as can be discerned, ␣1 mutations do not have an effect on assembly of gel-shif...

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“…Recombinant TFIIE-34 and TFIIE-56 were gifts from Mary Maxon and had been expressed in E. coli and purified as described (Peterson et al 1991). Recombinant RAP30 and RAP74 were expressed in E. coli and purified as described (Aso et al 1992;Finkelstein et al 1992). The hTFIID fraction (phosphocellulose, 1.0 M eluate) and hTFIIA fraction (phosphocellulose, 0.1 M flowthrough: DE-52, 0.35 M wash) were isolated as described previously (Reinberg et al 1987).…”

Section: In Vitro Transcriptionmentioning

confidence: 99%

Drosophila TFIIA-L is processed into two subunits that are associated with the TBP/TAF complex.

Yokomori

Admon²,

Goodrich³

et al. 1993

Genes Dev.

105

122

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The basal factor TFIIA has been shown to act early during initiation in both the mammalian and yeast transcription systems, but a TFIIA-like activity has not been identified in Drosophila. While characterizing the Drosophila TFIID complex, we discovered that a 30-kD protein that cofractionated with dTFIID was homologous to the previously identified, large subunit of yeast TFIIA. Here, we report the cloning and biochemical characterization of Drosophila TFIIA-L. Coimmunoprecipitation studies with anti-dTBP, anti-dTFIIA-L, and anti-TAF antibodies indicated a tight association of the endogenous dTFIIA and dTFIID. However, dTFIIA could be dissociated from dTFIID under conditions that did not elute the TAFs, and the eluted material had mobility shift and transcriptional activities associated with TFIIA. Peptide sequence and Western analysis with antibodies raised against the amino-and carboxy-terminal portions of recombinant dTFIIA-L revealed that a precursor 48-kD species was cleaved in vivo, giving rise to the 30-and 20-kD subunits of dTFIIA that remain associated with each other and with dTFIID. Protein-protein interaction assays identified dTBP and dTAFn110 as targets for binding TFIIA in the TFIID complex. These results suggest that TFIIA may form a specific complex with both TAFs and other components of the transcriptional machinery during formation of the initiation complex.

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Characterization of cDNA for the large subunit of the transcription initiation factor TFIIF

Cited by 80 publications

References 18 publications

Identification of a minimal set of proteins that is sufficient for accurate initiation of transcription by RNA polymerase II.

Identification of a minimal set of proteins that is sufficient for accurate initiation of transcription by RNA polymerase II.

A Key Role for the α1 Helix of Human RAP74 in the Initiation and Elongation of RNA Chains

Drosophila TFIIA-L is processed into two subunits that are associated with the TBP/TAF complex.

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