The subunit structure of Saccharomyces cerevisiae transcription factor IIIC probed with a novel photocrosslinking reagent.

Bartholomew, Blaine; Kassavetis, George A.; Braun, Burkhard R.; Geiduschek, E. Peter

doi:10.1002/j.1460-2075.1990.tb07389.x

Cited by 183 publications

(183 citation statements)

References 34 publications

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“…4 provides a framework for interpretation of genetic, biochemical, and structural data on RNAPII-dependent transcription initiation and regulation. Based on similarities between RNAPII and other multisubunit RNA polymerases (i.e., RNA polymerases I and III, E. coli RNA polymerase) in threedimensional structure (39,40), subunit primary structure (41,42), DNA crosslinking (43)(44)(45)(46)(47), DNA bending (48)(49)(50)(51), and DNA wrapping (51-54), we suggest that the model in Fig. 4 may apply generally to multisubunit RNA polymerases.…”

Section: Discussionmentioning

confidence: 92%

Trajectory of DNA in the RNA polymerase II transcription preinitiation complex

Kim

Lagrange

Wang

et al. 1997

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

111

124

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By using site-specific protein-DNA photocrosslinking, we define the positions of TATA-binding protein, transcription factor IIB, transcription factor IIF, and subunits of RNA polymerase II (RNAPII) relative to promoter DNA within the human transcription preinitiation complex. The results indicate that the interface between the largest and second-largest subunits of RNAPII forms an extended, Ϸ240 Å channel that interacts with promoter DNA both upstream and downstream of the transcription start. By using electron microscopy, we show that RNAPII compacts promoter DNA by the equivalent of Ϸ50 bp. Together with the published structure of RNAPII, the results indicate that RNAPII wraps DNA around its surface and suggest a specific model for the trajectory of the wrapped DNA.Transcription initiation at a eukaryotic protein-encoding gene involves assembly on promoter DNA of a complex consisting of RNA polymerase II (RNAPII) and six general transcription factors: IIA, IIB, IID (or TATA-element binding protein, TBP), IIE, IIF, and IIH (1-3). A subcomplex containing TBP, IIB, IIF, RNAPII, and promoter DNA is stable (4, 5), and, under certain conditions (e.g., conditions that promote DNA melting), is fully competent for transcription initiation (6-11). Results of DNA footprinting experiments indicate that the TBP-IIB-IIF-RNAPII-promoter complex with linear DNA at 30°C involves interactions both upstream and downstream of the transcription start (positions Ϫ42 to ϩ17; H. Lu and D.R., unpublished data) and involves unmelted DNA (11). Thus, the TBP-IIB-IIF-RNAPII-promoter complex with linear DNA at 30°C appears to correspond to the RNA polymerase-promoter ''intermediate complex'' characterized in studies of Escherichia coli RNA polymerase (RP i or RP c2 ; refs. 12-15).The TBP-IIB-IIF-RNAPII-promoter complex contains at least 14 distinct polypeptides (one in TBP, one in IIB, two in IIF, and at least 10 in RNAPII) and has a molecular mass in excess of 700 kDa (1-3). High-resolution structures have been determined for the TBP-DNA and TBP-IIB-DNA complexes (16-18). However, the TBP-IIB-IIF-RNAPII-promoter complex is too large for high-resolution structure determination by current methods. Therefore, information about the structure of the TBP-IIB-IIF-RNAPII-promoter complex must rely on low-resolution structure determination (19,20) supplemented by biochemical and imaging data.In the work in this report, we have used site-specific protein-DNA photocrosslinking and electron microscopy to define protein-DNA interactions within the human TBP-IIB-IIF-RNAPIIpromoter complex. MATERIALS AND METHODSDerivatized Promoter DNA Fragments. Derivatized promoter DNA fragments were prepared essentially as in ref. 21. Oligodeoxyribonucleotides containing phosphorothioate 5Ј to the third nucleotide were synthesized by using solid-phase ␤-cyanoethylphosphoramidite chemistry and tetraethylthiuram disulfide (Applied Biosystems), purified on OPC (Applied Biosystems), and derivatized with azidophenacyl bromide (Sigma; ref. 22). Derivatized oligodeo...

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Section: Discussionmentioning

confidence: 92%

Trajectory of DNA in the RNA polymerase II transcription preinitiation complex

Kim

Lagrange

Wang

et al. 1997

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

111

124

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“…The DNA repair pre-incision complex containing either XPC/HR23B alone or XPC/HR23B and TFIIH was incubated with the various photoprobes in the presence of ATP and further UV-irradiated. Under those conditions, the photoreactive nitrene of AB-dUMP can be covalently crosslinked to the polypeptides located at a distance of ϳ10 A from the DNA backbone (40,41,59). The samples were then digested with exonucleases to remove non-crosslinked DNA tails, and the crosslinked polypeptides were analyzed by SDS-PAGE.…”

Section: Resultsmentioning

confidence: 99%

Ordered Conformational Changes in Damaged DNA Induced by Nucleotide Excision Repair Factors

Tapias

Auriol

Forget

et al. 2004

Journal of Biological Chemistry

142

152

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In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position ؊3 to ؉6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position ؊6 to ؉6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position ؊19 to ؉8. XPG and XPF/ERCC1 endonucleases then cut the damaged DNA at the limit of the opened structure that was previously "labeled" by the positioning of XPC/HR23B and TFIIH.To counteract the detrimental effect of genotoxic attacks, cells activate sophisticated and specific DNA repair pathways. Damage induced by UV radiation, environmental agents, and anticancer drugs are removed by two distinct nucleotide excision repair (NER) 1 subpathways, namely global genome repair (GGR), which eliminates lesions from the entire genome, and transcription-coupled repair (TCR), a specialized pathway that repairs damages on a transcribed strand of active genes (1-3). Human NER involves the ordered action of factors in dual incision and DNA repair resynthesis steps (4). Any mutation that affects either the enzymatic activity or the ordered assembly of the dual incision complex leads to genetic disorders such as xeroderma pigmentosum, trichothiodystrophy, or Cockayne syndrome (5, 6).In global genome repair, the dual incision is a multistep process that results from the coordinated action of XPC/ HR23B, TFIIH, XPA, RPA, XPG, and XPF/ERCC1, resulting in the removal of the damaged oligonucleotide (4, 7, 8). After being recognized by the XPC/HR23B complex, the damaged DNA structure is targeted by TFIIH, which recruits the other factors upon the addition of ATP (9 -11). The unwound DNA is then incised by the two endonucleases XPG and XPF/ERCC1 on the 3Ј and 5Ј side of the lesion, respectively (12-15), leaving a gap structure that is filled up by the DNA polymerase ⑀ or ␦ and the accompanying factors PCNA, RF-C, RPA, and DNA ligase I (16). Whether or not the NER reaction occurs by sequential arrival of the various factors or by a pre-assembled complex referred to as the repairosome or the holoenzyme is still under debate (17)(18)(19). Although the hypothesis of the sequential assembly, which has gained a lot of support from recent biological studies, seems to be more accepted, the order of assembly of the NER factors on the damaged DNA and their contribution to the DNA remodeling to allow the repair are not fully understood (10,20,21). As an example, to further learn abou...

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“…To determine which polypeptide (or polypeptides) of TFIID was in close proximity to the DPE (and, hence, likely to interact directly with the DPE), we carried out photo-cross-linking experiments with core promoter probes containing the thymidine analog 5-[N-(p-azidobenzoyl)-3-amino allyl]-deoxyuridine 5Ј-monophosphate (N 3 RdUMP; Bartholomew et al 1990Bartholomew et al , 1991Bartholomew et al , 1995. N 3 RdUMP has a photoreactive aryl azide moiety on a 9-to 10-Å tether that probes the space in the vicinity of the DNA major groove (Bartholomew et al 1990), and it has been used extensively in the study of proteins that interact with DNA. For instance, in the context of these studies, it is pertinent to note that N 3 RdUMP was used in the analysis of the binding of human TFIID to the TATA-containing, DPE-less adenovirus major late promoter .…”

Section: Alteration Of the Dpe Sequence Does Not Affect Transcript Stmentioning

confidence: 99%

“…George Kassavetis and E. Peter Geiduschek (University of California, San Diego, La Jolla). The N 3 RdUTP was synthesized and used as described previously (Bartholomew et al 1990(Bartholomew et al , 1995, except that the DNA probes were prepared from synthetic oligonucleotides instead of single-stranded M13 DNA. The specific DNA sequences and reaction conditions are available upon request.…”

Section: Uv Cross-linkingmentioning

confidence: 99%

The downstream core promoter element, DPE, is conserved fromDrosophila to humans and is recognized by TAF_II60 of Drosophila

Burke

Kadonaga²

1997

Genes Dev.

462

379

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We analyzed the function of the downstream promoter element (DPE), a distinct 7-nucleotide core promoter element that is ∼30 nucleotides downstream of the transcription start site of many TATA-box-deficient (TATA-less) promoters in Drosophila. There is a strict requirement for spacing between the Inr and DPE motifs, as an increase or decrease of 3 nucleotides in the distance between the Inr and DPE causes a seven-to eightfold reduction in transcription as well as a significant reduction in the binding of purified TFIID. These results suggest a specific and somewhat rigid interaction of TFIID with the Inr and DPE sequences. Photo-cross-linking analysis of purified TFIID with a TATA-less DPE-containing promoter revealed specific cross-linking of dTAF II 60 and dTAF II 40 to the DPE, with a higher efficiency of cross-linking to dTAF II 60 than to dTAF II 40. These data, combined with the previously well-characterized interactions between the two TAFs and their homology to histones H4 and H3, suggest that a dTAF II 60-dTAF II 40 heterotetramer binds to the DPE. Human and Drosophila transcription factors exhibit essentially the same requirements for DPE sequence and for Inr-DPE spacing. In addition, the TATA-less promoter of the human interferon regulatory factor-1 (IRF-1) gene contains a DPE that is important for transcriptional activity both in vitro and in cultured cells. Hence, these studies provide evidence for a direct role of TAFs in basal transcription of TATA-less DPE-containing genes and collectively indicate that the DPE is, in many respects, a downstream counterpart to the TATA box that is present in Drosophila to humans. Transcription is centrally involved in an array of biological processes, which include growth, development, and response to external stimuli. In eukaryotes, protein-coding genes are transcribed by the RNA polymerase II transcriptional machinery, which comprises RNA polymerase II and other factors that are required for basal and regulated transcription. Transcription by RNA polymerase II is directed by cis-acting DNA sequences that typically consist of a core promoter along with regulatory elements, such as enhancers, that contain binding sites for sequence-specific transcriptional activator and/or repressor proteins. Thus, the study of both the trans-acting protein factors and the cis-acting DNA elements is necessary to gain a better understanding of the fundamental mechanisms by which genes are transcribed (for recent reviews, see Bjö rkland and

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The subunit structure of Saccharomyces cerevisiae transcription factor IIIC probed with a novel photocrosslinking reagent.

Cited by 183 publications

References 34 publications

Trajectory of DNA in the RNA polymerase II transcription preinitiation complex

Trajectory of DNA in the RNA polymerase II transcription preinitiation complex

Ordered Conformational Changes in Damaged DNA Induced by Nucleotide Excision Repair Factors

The downstream core promoter element, DPE, is conserved fromDrosophila to humans and is recognized by TAF_II60 of Drosophila

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The subunit structure of Saccharomyces cerevisiae transcription factor IIIC probed with a novel photocrosslinking reagent.

Cited by 183 publications

References 34 publications

Trajectory of DNA in the RNA polymerase II transcription preinitiation complex

Trajectory of DNA in the RNA polymerase II transcription preinitiation complex

Ordered Conformational Changes in Damaged DNA Induced by Nucleotide Excision Repair Factors

The downstream core promoter element, DPE, is conserved fromDrosophila to humans and is recognized by TAFII60 of Drosophila

Contact Info

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The downstream core promoter element, DPE, is conserved fromDrosophila to humans and is recognized by TAF_II60 of Drosophila