TFIIH XPB mutants suggest a unified bacterial-like mechanism for promoter opening but not escape

Lin, Yin; Choi, Wai; Gralla, Jay D.

doi:10.1038/nsmb949

Cited by 76 publications

(77 citation statements)

References 37 publications

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“…For 54 , ATP-dependent activator proteins are hypothesized to remodel the downstream jaw by means of interactions with the NTD of 54 . Similarly, ATP hydrolysis by the transcription factor TFIIH has been proposed to drive large-scale protein rearrangements in the pol II preinitiation complex to allow DNA melting (30). This hypothesis contrasts with the generally accepted model that invokes ATP-driven conformational changes in the helicase domains of TFIIH to open DNA (4).…”

Section: Comparison Of the Extent Of Dna Opening In Rpo With I1 Usingmentioning

confidence: 99%

“…At 17°C, where the equilibrium constant K 1 is a maximum and conversion of I 1 to I 2 is relatively slow, we previously found that the fraction of promoter DNA in I 1 complexes ( I1 ) at 15 sec after mixing P R promoter with 70 nM (excess) RNAP was 0.55 (6). To increase the fraction of I 1 in the time interval (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) (38) where the only complex predicted to be populated is I 1 (9). Because the signal:noise ratio in these 0°C experiments was marginal, and to avoid the possibility of populating off-pathway complexes at 0°C (39), we identified solution conditions where I 1 is Ͼ70% of promoter DNA (see above) at early times in the time course of RP o formation, and footprinted as follows.…”

Section: Determination Of Fractions Of Promoter Dna In I1 and Rpo Commentioning

confidence: 99%

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Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

Davis¹,

Bingman

Landick³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

197

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initiation ͉ transcription ͉ wrapping ͉ kinetics S pecific transcription initiation by Escherichia coli RNA polymerase (RNAP: core subunit composition ␣ 2 ␤␤Ј ϩ 70 ϭ holoenzyme) at promoter sequences is determined by recognition of DNA (Ϫ10 and Ϫ35 hexamers) upstream of the start site (ϩ1) by the specificity subunit 70 . Subsequent to binding, a series of large-scale conformational changes in both RNAP and promoter DNA create the initiation-competent open complex (RP o ) (1). During these steps, the multisubunit bacterial RNAP acts as an intricate molecular machine and opens Ϸ14 bp of the DNA double helix. Defining the cascade of conformational changes that occur during initiation is essential to understand sequence-and factordependent regulation of the rate of transcription initiation and has important applications in chemical biology and in antibiotic design. However, the intermediates on this pathway are relatively unstable and short-lived and hence are difficult to trap unambiguously. To date, all structural information about complexes known to be on-pathway intermediates in RP o formation has come from chemical and enzymatic DNA footprinting methods.Quantitative kinetic-mechanistic studies find that at least two kinetically significant intermediates, generically designated I 1 and I 2 , precede formation of RP o by E. coli RNAP:where the relatively slow interconversions between I 1 and I 2 are rate-limiting in both the forward and back directions (2, 3). In the mechanism shown in Eq. 1, I 2 and RP o are characterized by their resistance to a short challenge with a polyanionic competitor such as heparin, which acts to sequester any free RNAP present during the challenge. (In contrast, after a 10 to 20 sec challenge with heparin, I 1 complexes, which are in rapid equilibrium with free RNAP and promoter sequences, are eliminated from the population.) Given the high degree of conservation of bacterial RNAP and promoter DNA sequences, this mechanism is likely to describe the key steps in initiation in most prokaryotes. Moreover, conservation of many elements of sequence, structure, and/or function between bacterial and eukaryotic polymerase (pol II) subunits and transcription factors supports the inference that the bacterial intermediates may be homologs of initiation intermediates formed by pol II (4, 5).Recently we (6) and Ross and Gourse (7) found that the presence of DNA upstream of the Ϫ35 promoter recognition hexamer greatly accelerates (up to Ϸ60-fold) the rate-determining isomerization step (conversion of I 1 to I 2 ). Strikingly, DNase I footprinting of I 1 at the strong bacteriophage promoter P R reveals that when nonspecific DNA upstream of base pair Ϫ47 is present, downstream DNA is protected to around ϩ20, and thus bound in the active-site channel of RNAP. However, when DNA upstream of Ϫ47 is deleted,

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Section: Comparison Of the Extent Of Dna Opening In Rpo With I1 Usingmentioning

confidence: 99%

Section: Determination Of Fractions Of Promoter Dna In I1 and Rpo Commentioning

confidence: 99%

Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

Davis¹,

Bingman

Landick³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

197

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“…Biochemical and genetic studies have shown that both XPB and XPD ATPase activities are needed to open up DNA around a damaged site (18,19). Recent data showed that only XPB ATPase activity of is required for opening and remodeling of DNA in NER and transcription, and its helicase is devoted to promoter escape in transcription (20,21). XPD helicase activity, on the other hand, plays a minor role in transcription but is necessary for NER (19,22).…”

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confidence: 99%

ARCH domain of XPD, an anchoring platform for CAK that conditions TFIIH DNA repair and transcription activities

Abdulrahman

Iltis

Radu

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The xeroderma pigmentosum group D (XPD) helicase is a subunit of transcription/DNA repair factor, transcription factor II H (TFIIH) that catalyzes the unwinding of a damaged DNA duplex during nucleotide excision repair. Apart from two canonical helicase domains, XPD is composed of a 4Fe-S cluster domain involved in DNA damage recognition and a module of uncharacterized function termed the "ARCH domain." By investigating the consequences of a mutation found in a patient with trichothiodystrophy, we show that the ARCH domain is critical for the recruitment of the cyclin-dependent kinase (CDK)-activating kinase (CAK) complex. Indeed, this mutation not only affects the interaction with the MAT1 CAK subunit, thereby decreasing the in vitro basal transcription activity of TFIIH itself and impeding the efficient recruitment of the transcription machinery on the promoter of an activated gene, but also impairs the DNA unwinding activity of XPD and the nucleotide excision repair activity of TFIIH. We further demonstrate the role of CAK in downregulating the XPD helicase activity within TFIIH. Taken together, our results identify the ARCH domain of XPD as a platform for the recruitment of CAK and as a potential molecular switch that might control TFIIH composition and play a key role in the conversion of TFIIH from a factor active in transcription to a factor involved in DNA repair.rare disease | regulation of gene expression T he xeroderma pigmentosum group D (XPD) gene encodes a 5′-3′ helicase (XPD) that harbors mutations in patients suffering from three rare autosomal recessive diseases, xeroderma pigmentosum (XP), trichothiodystrophy (TTD), and Cockayne syndrome (CS) (1, 2). XP is characterized by a deficit of the nucleotide excision repair (NER) pathway, leading to sun sensitivity and susceptibility to skin cancer. TTD is characterized by sulfur-deficient brittle hair and a variety of neuroectodermal symptoms (3). XPD is the founding member of a family of DNA helicases conserved in archaea and eukaryotes. All family members share a four-domain organization including a conserved (Fe-S) cluster-binding domain that is essential for the helicase activity and a module of uncharacterized function named the ARCH domain by its arch-shape structure (4-7). Although archeal XPD homologs are monomers and have no known stable interactors, eukaryotic XPD homologs are part of the general transcription/DNA repair factor transcription factor II H (TFIIH), a multisubunit complex made up of 10 subunits (reviewed in ref. 8). Low-resolution models for TFIIH have been obtained for the complex in yeast (9, 10) and for the human complex (11), showing an overall conservation of shape. Human TFIIH can be resolved into two functional and structural entities bridged by XPD: the core-TFIIH consists of XPB, p62, p52, p44, p34, and p8, whereas the cyclin-dependent kinase (CDK)-activating kinase (CAK) subcomplex contains CDK7, cyclin H, and ménage a trois 1 (MAT1). XPD interacts with the p44 core-TFIIH subunit and with MAT1, a subunit of CAK involve...

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“…In both cases, passing from the closed to the open RNAP promoter complex requires activator proteins that hydrolyze nucleoside triphosphate to drive open RNAP promoter complex formation (3). The 54 -RNAP binds to specific promoter sites centered on positions Ϫ24 and Ϫ12 relative to the transcription start site and remains in a transcriptionally silent conformation.…”

mentioning

confidence: 99%

Sensor I Threonine of the AAA+ ATPase Transcriptional Activator PspF Is Involved in Coupling Nucleotide Triphosphate Hydrolysis to the Restructuring of σ54-RNA Polymerase

Schumacher

Joly

Rappas

et al. 2007

Journal of Biological Chemistry

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Transcriptional initiation invariably involves the transitionRegulation of transcription enables cells to adapt and differentiate through coordination of protein synthesis. Two major mechanisms exist to control gene transcription as follows: one through recruitment to or preventing the RNA polymerase (RNAP) 4 binding to promoter DNA and one through activation or inhibition of the RNA polymerase activity. Despite the variations in subunit numbers that constitute the basal transcription machinery, RNA polymerases are structurally and functionally highly conserved in all kingdoms of life (1). During the initiation of transcription, RNA polymerases invariably melt the promoter DNA and have to engage the single-stranded template DNA in the structurally conserved catalytic cleft (2).The major variant bacterial 54 -RNAP is regulated by an activating mechanism with some resemblance to the operation of eukaryotic RNAP II. In both cases, passing from the closed to the open RNAP promoter complex requires activator proteins that hydrolyze nucleoside triphosphate to drive open RNAP promoter complex formation (3). The 54 -RNAP binds to specific promoter sites centered on positions Ϫ24 and Ϫ12 relative to the transcription start site and remains in a transcriptionally silent conformation. Open complexes of the 54 -RNAP promoter complexes are thermodynamically labile, and activation relies on the productive coupling of nucleoside triphosphate hydrolysis (energy coupling) from 54 activators to the 54 -RNAP promoter complex. The 54 activator-dependent initial events in open complex formation are mediated by structural changes between 54 and the promoter DNA, which can be melted from position Ϫ12 to Ϫ5 in a reaction that can occur independently from RNAP core determinants. The restructuring of promoter DNA by activated 54 has been termed 54 isomerization (4 -6). Following the initial events and requiring the presence of the RNAP core, the promoter DNA opening extends to position ϩ3 relative to the transcription start site (7).Energy coupling primarily involves intimate interactions between the activator and the 54 promoter complex. 54 activators, also termed enhancer-binding proteins (EBP), belong to the versatile AAAϩ protein (ATPases associated with various cellular activities) family of molecular machines (for review see Refs. 8 -10). Members of EBPs include the well studied NtrC, PspF, DctD, XylR, NifA, and DmpR proteins (11). PspF is the phage shock protein F that activates transcription of psp genes involved in the phage shock response in Escherichia coli (12, * This work was supported in part by the Biotechnology and Biological Sciences Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence may be addressed. Tel.: 44-207-5945366; Fax: 44-207-5842056; E-mail: j.schumacher@imperial.ac.uk. 2 Recipient of EMBO F...

show abstract

TFIIH XPB mutants suggest a unified bacterial-like mechanism for promoter opening but not escape

Cited by 76 publications

References 37 publications

Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

ARCH domain of XPD, an anchoring platform for CAK that conditions TFIIH DNA repair and transcription activities

Sensor I Threonine of the AAA+ ATPase Transcriptional Activator PspF Is Involved in Coupling Nucleotide Triphosphate Hydrolysis to the Restructuring of σ54-RNA Polymerase

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