Mechanism of Promoter Melting by the Xeroderma Pigmentosum Complementation Group B Helicase of Transcription Factor IIH Revealed by Protein-DNA Photo-Cross-Linking

Douziech, Maxime; Coin, Frédéric; Chipoulet, Jean-Marc; Arai, Yoko; Ohkuma, Yoshiaki; Egly, Jean‐Marc; Coulombe, Benoit

doi:10.1128/.20.21.8168-8177.2000

Cited by 4 publications

(2 citation statements)

References 37 publications

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“…With the upstream end of the pol II–DNA complex rotationally fixed through its interaction with TFIIB and TBP, the twist deficit translocates to, and initiates the opening of, the transcription bubble. The second model (Douziech et al ., 2000) invokes a wrapping and peeling mechanism in which TFIIH tightens the wrapping of DNA around the promoter complex, somehow induces localized DNA unwinding near the start site of transcription and utilizes the XPB helicase to complete ATP hydrolysis‐dependent promoter opening.…”

Section: Discussionmentioning

confidence: 99%

Marking the start site of RNA polymerase III transcription: the role of constraint, compaction and continuity of the transcribed DNA strand

et al. 2002

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The effects of breaks in the individual strands of an RNA polymerase III promoter on initiation of transcription have been examined. Single breaks have been introduced at 2 bp intervals in a 24 bp segment that spans the transcriptional start site of the U6 snRNA gene promoter. Their effects on transcription are asymmetrically distributed: transcribed (template) strand breaks downstream of bp ±14 (relative to the normal start as +1) systematically shift the start site, evidently by disrupting the normal mechanism that measures distance from DNA-bound TBP. Breaks placed close to the normal start site very strongly inhibit transcription. Breaks in the non-transcribed strand generate only minor effects on transcription. A structure-based model interprets these observations and explains how the transcribed strand is used to locate the transcriptional start site. Keywords: RNA polymerase III/S.cerevisiae/TFIIIB/ transcriptional initiation/U6 promoter Introduction Yeast RNA polymerase (pol) III is brought to its promoters primarily by interactions with its central transcription initiation factor, TFIIIB. TFIIIB is composed of three subunits: TBP, Brf1 and Bdp1. (Brf1 was called Brf, and Bdp1 was called B¢¢ in previous work from this laboratory. See Materials and methods for a note on the new nomenclature.) Although TBP secures attachment of TFIIIB to strong TATA boxes, most budding yeast (Saccharomyces cerevisiae) pol III-transcribed genes do not have strong TATA boxes; recruitment of TFIIIB to their promoters is secured by TFIIIC, which binds to the gene-internal boxA and boxB promoter elements. On 5S rRNA genes, TFIIIC attaches to a transcription unitinternal protein platform created by TFIIIA. The connection between TFIIIB and TFIIIC is cemented primarily by their respective Brf1 and Tfc4 subunits (reviewed by White, 1998). Brf1 derives its acronym (for TFIIB-Related Factor) from the homology of its N-proximal half with the pol II transcription factor TFIIB; the C-proximal half contains determinants primarily responsible for holding TFIIIB together through a strong interaction with TBP and a somewhat weaker interaction with Bdp1, the third subunit of TFIIIB (Kassavetis et al., 1998a).TBP kinks sharply and bends DNA, and Bdp1 introduces an additional DNA bend downstream of the TATA box (Grove et al., 1999). It is the complexly bent TFIIIB±DNA complex that brings budding yeast pol III directly to the promoter (Kassavetis et al., 1990). The principal interactions securing pol III recruitment involve Brf1 and the C34 subunit of pol III (Brun et al., 1997;Andrau et al., 1999). (C34 is part of a three-protein pol III subassembly that is speci®cally required for transcription initiation.) Additional pol III±TFIIIB interactions involve Brf1 and the C17 subunit of pol III (Che Âdin et al., 1998;Flores et al., 1999;Ferri et al., 2000; reviewed by White, 1998;Geiduschek and Kassavetis, 2001). TFIIIB also plays an essential post-recruitment role in initiation of transcription by pol III by guiding promoter opening. This role h...

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Section: Discussionmentioning

confidence: 99%

Marking the start site of RNA polymerase III transcription: the role of constraint, compaction and continuity of the transcribed DNA strand

et al. 2002

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“…In RNA and DNA, local structural fluctuations involving the opening and closing of basepairs occur frequently. This local melting is of functional importance to many processes, including transcription, replication, and various DNA repair mechanisms (1)(2)(3)(4)(5)(6)(7). A delicate balance is required between structural integrity in the basepaired state and the flexibility to transiently populate an open state to enable information read-out.…”

Section: Introductionmentioning

confidence: 99%

Individual Basepair Stability of DNA and RNA Studied by NMR-Detected Solvent Exchange

Steinert

Rinnenthal

Schwalbe

2012

Biophysical Journal

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In this study, we have optimized NMR methodology to determine the thermodynamic parameters of basepair opening in DNA and RNA duplexes by characterizing the temperature dependence of imino proton exchange rates of individual basepairs. Contributions of the nuclear Overhauser effect to exchange rates measured with inversion recovery experiments are quantified, and the influence of intrinsic and external catalysis exchange mechanisms on the imino proton exchange rates is analyzed. Basepairs in DNA and RNA have an approximately equal stability, and the enthalpy and entropy values of their basepair dissociation are correlated linearly. Furthermore, the compensation temperature, T(c), which is derived from the slope of the correlation, coincides with the melting temperature, and duplex unfolding occurs at that temperature where all basepairs are equally thermodynamically stable. The impact of protium-deuterium exchange of the imino hydrogen on the free energy of RNA basepair opening is investigated, and it is found that two A·U basepairs show distinct fractionation factors.

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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

Watanabe

Hayashi

Tanaka

et al. 2003

Molecular and Cellular Biology

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The general transcription factor TFIIE plays essential roles in both transcription initiation and the transition from initiation to elongation. Previously, we systematically deleted the structural motifs and characteristic sequences of the small subunit of human TFIIE (hTFIIE␤) to map its functional regions. Here we introduced point mutations into two regions located near the carboxy terminus of hTFIIE␤ and identified the functionally essential amino acid residues that bind to RNA polymerase II (Pol II), the general transcription factors, and single-stranded DNA. Although most residues identified were essential for transcription initiation, use of an in vitro transcription assay with a linearized template revealed that several residues in the carboxyterminal helix-loop region are crucially involved in the transition stage. Mutations in these residues also affected the ability of hTFIIE␤ to stimulate TFIIH-mediated phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of Pol II. Furthermore, these mutations conspicuously augmented the binding of hTFIIE␤ to the p44 subunit of TFIIH. The antibody study indicated that they thus altered the conformation of one side of TFIIH, consisting of p44, XPD, and Cdk-activating kinase subunits, that is essential for the transition stage. This is an important clue for elucidating the molecular mechanisms involved in the transition stage.In eukaryotes, the expression of protein-coding genes is strictly regulated at the level of transcription by RNA polymerase II (Pol II). Once signals from outside the nucleus are received and the condensed form of the inactive chromatin is activated and remodeled by chromatin-modulating factors, five general transcription factors (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) together with Pol II form the preinitiation complex (PIC) on the core promoter. Formation of this complex is assisted by various transcriptional activators, cofactors, and mediators (for reviews, see references 19, 25, and 43). Analyses of the PIC assembly pathway using isolated general transcription factors have revealed that the factors can assemble stepwise in vitro. This process commences with the binding of TFIID to the TATA box on the core promoter and ends with TFIIE and TFIIH joining the PIC (reviewed in references 10, 26, 34, and 42). It is widely accepted that TFIIE and TFIIH stabilize and activate the PIC by binding to all the other general transcription factors as well as to Pol II and at the same time open up the double-stranded DNA (dsDNA) at the region from Ϫ9 to ϩ2, adjacent to the transcription initiation site (ϩ1), in a manner that is dependent on dATP hydrolysis (14,56). This process is known as promoter melting. These various functions of TFIIE and TFIIH have been revealed recently by three types of studies. First, photo-cross-linking studies demonstrated that TFIIE␤ binds directly to the core promoter region between positions Ϫ14 and Ϫ2, which is where the promoter melts upon transcription initiation (5, 41). Second, two-dimensiona...

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Mechanism of Promoter Melting by the Xeroderma Pigmentosum Complementation Group B Helicase of Transcription Factor IIH Revealed by Protein-DNA Photo-Cross-Linking

Cited by 4 publications

References 37 publications

Marking the start site of RNA polymerase III transcription: the role of constraint, compaction and continuity of the transcribed DNA strand

Marking the start site of RNA polymerase III transcription: the role of constraint, compaction and continuity of the transcribed DNA strand

Individual Basepair Stability of DNA and RNA Studied by NMR-Detected Solvent Exchange

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

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