The Carboxy Terminus of the Small Subunit of TFIIE Regulates the Transition from Transcription Initiation to Elongation by RNA Polymerase II

Watanabe, Tomomichi; Hayashi, Katsuhiko; Tanaka, Aki; Furumoto, Tadashi; Hanaoka, Fumio; Ohkuma, Yoshiaki

doi:10.1128/mcb.23.8.2914-2926.2003

Cited by 25 publications

(54 citation statements)

References 62 publications

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“…Although TFIIE has no catalytic activities (14,15), it can influence those of TFIIH (16,24,36,38). It has been reported to positively regulate the ATPase and kinase activities of TFIIH (19) and negatively regulate its helicase activity (39).…”

Section: Introductionmentioning

confidence: 99%

Stimulation of the XPB ATP-dependent helicase by the beta subunit of TFIIE

Lin

Gralla

2005

Nucleic Acids Research

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TFIIE and TFIIH are essential for the promoter opening and escape that occurs as RNA polymerase II transits into early elongation. XPB, a subunit of TFIIH, contains an ATP-dependent helicase activity that is used in both of these processes. Here, we show that the smaller beta subunit of TFIIE stimulates the XPB helicase and ATPase activities. The larger alpha subunit can use its known inhibitory activity to moderate the stimulation by the beta subunit. Regions of TFIIE beta required for the helicase stimulation were identified. Mutants were constructed that are defective in stimulating the XPB helicase but still allow intact TFIIE to bind and recruit XPB and TFIIH to form the pre-initiation complex. In a test for the functional significance of the stimulatory effect of TFIIE beta, these mutant forms of TFIIE were shown to be defective in a transcription assay on linear DNA. The data suggest that the beta subunit of TFIIE is an ATPase and helicase co-factor that can assist the XPB subunit of TFIIH during transcription initiation and the transition to early elongation, enhancing the potential diversity of regulatory targets.

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Section: Introductionmentioning

confidence: 99%

Stimulation of the XPB ATP-dependent helicase by the beta subunit of TFIIE

Lin

Gralla

2005

Nucleic Acids Research

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“…In vitro transcription with either a supercoiled or a linearized template was carried out with increasing amounts (12 and 24 ng) of wild type or mutant hTFIIE␣ proteins. The plasmid pML(C 2 AT)⌬-50, which contains the adenovirus 2 major late promoter and gives a 390-nucleotide transcript, was used as a template (31). To prepare the linearized template, pML(C 2 AT)⌬-50 was digested with SmaI.…”

Section: Methodsmentioning

confidence: 99%

“…GST-Pull-down Assay-GST fusion proteins were performed as previously described (31). The bound proteins were detected by Western blotting with anti-hTFIIE␣ mouse monoclonal antibody (1:8,000 dilution) (Medical Biology Laboratory).…”

Section: Methodsmentioning

confidence: 99%

“…In Vitro Transcription Assays-Recombinant general transcription factors as well as native pol II and TFIIH were purified as described previously (31). In vitro transcription with either a supercoiled or a linearized template was carried out with increasing amounts (12 and 24 ng) of wild type or mutant hTFIIE␣ proteins.…”

Section: Methodsmentioning

confidence: 99%

“…Structures were analyzed and displayed using PROCHECK-NMR (28), GRASP (29), MOLMOL (30), and SYBYL (Tripos Inc., St. Louis, MO). (31). A restriction enzyme site was placed in an oligonucleotide to select for properly mutated plasmids as described elsewhere, and the mutants were then checked by sequencing using an ABI Prism 310 Genetic Analyzer (Applied Biosystems).…”

Section: Methodsmentioning

confidence: 99%

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A Novel Zinc Finger Structure in the Large Subunit of Human General Transcription Factor TFIIE

Okuda

Tanaka

Arai

et al. 2004

Journal of Biological Chemistry

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The zinc finger domain in the large subunit of TFIIE (TFIIE␣) is phylogenetically conserved and is essential for transcription. Here, we determined the solution structure of this domain by using NMR. It consisted of one ␣-helix and five ␤-strands, showing novel features distinct from previously determined zinc-binding structures. We created point mutants of TFIIE␣ in this domain and examined their binding abilities to other general transcription factors as well as their transcription activities. Four Zn 2؉ -ligand mutants, in which each of cysteine residues at positions 129, 132, 154, and 157 was replaced by alanine, possessed no transcription activities on a linearized template, whereas, on a supercoiled template, interesting functional asymmetry was observed: although the C-terminal two mutants abolished transcription activity (<5%), the N-terminal two mutants retained about 20% activities. The N-terminal two mutants bound stronger to the small subunit of TFIIF than the wild type and the C-terminal two mutants were impaired in their binding abilities to the XPB subunits of TFIIH. These suggest that the structural integrity of the zinc finger domain is essential for the TFIIE function, particularly in the transition from the transcription initiation to elongation and the conformational tuning of this domain for appropriate positioning of TFIIF, TFIIH, and polymerase II would be needed depending on the situation and timing.In eukaryotes, transcription initiation of protein-coding genes requires RNA polymerase II (pol II) 1 and its auxiliary five general transcription factors, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (1, 2). Pol II and general transcription factors assemble together on a promoter DNA to form a preinitiation complex (PIC). TATA box-binding protein, a DNA binding subunit of TFIID, binds first to the TATA box. TFIIB then binds and recruits pol II to the complex (3). Pol II comes with TFIIF, which is important for accurate location of pol II in the complex around the transcription initiation site (4). Finally, TFIIE and TFIIH are incorporated to complete PIC formation (3).During PIC formation, TFIIE interacts with various general transcription factors, pol II, and promoter DNA, recruits TFIIH into the PIC, and regulates the enzymatic activities of TFIIH; serine kinase of the C-terminal domain of the largest subunit of pol II, DNA-dependent ATPase, and DNA helicase activities (5-7). At transcription initiation, TFIIE binds both to pol II near its active site and to promoter DNA ϳ10 bp upstream (Ϫ10) from the transcription initiation site (ϩ1), where the promoter melting starts (8). There TFIIE conducts pol II to sit at the initiation site, makes pol II processive upon C-terminal domain phosphorylation by stimulating C-terminal domain kinase activity of TFIIH, and simultaneously assists a helicase subunit XPB of TFIIH to start melting at the Ϫ10 position. At the transition from initiation to elongation, TFIIE also plays a direct role in promoter clearance by regulating kinase and DNA helicase activities o...

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The Carboxy Terminus of the Small Subunit of TFIIE Regulates the Transition from Transcription Initiation to Elongation by RNA Polymerase II

Cited by 25 publications

References 62 publications

Stimulation of the XPB ATP-dependent helicase by the beta subunit of TFIIE

Stimulation of the XPB ATP-dependent helicase by the beta subunit of TFIIE

A Novel Zinc Finger Structure in the Large Subunit of Human General Transcription Factor TFIIE

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