Electron Crystal Structure of an RNA Polymerase II Transcription Elongation Complex

Poglitsch, Claudia L.; Meredith, Gavin; Gnatt, Averell; Jensen, Grant J.; Chang, Wen Ho; Fu, Jiyang; Kornberg, Roger D.

doi:10.1016/s0092-8674(00)81513-5

Cited by 77 publications

(30 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The central core domain of TFIIE34 was recently shown to also contain a winged helix motif (43). The structure of the RNAPII elongation complex has revealed an important DNA bend in the region of the TIS (47). Second, RNAPII was found to crosslink to the promoter DNA from nucleotides Ϫ39/Ϫ40 to ϩ13 in the absence of TFIIH (51) and from nucleotides Ϫ55 to ϩ32/ϩ34 in the presence of TFIIH (this report).…”

Section: Topological Organization Of a Tbp-tfiib-tfiif-rnapii-tfiie-tmentioning

confidence: 77%

“…TBP binds to TFIIB and TFIIE; TFIIB binds to TFIIE and TFIIF; RNAPII binds to TBP, TFIIE, TFIIF, and TFIIH; TFIIF binds to TFIIE; and TFIIE binds to TFIIH. The structure of RNAPII has been modeled to account for both the dimension of the yeast enzyme and the presence of a 2.5-nm channel that can accommodate the promoter DNA and can exist in either an open or closed conformation (7,9,47,72). Finally, the structure of TFIIH is according to electron microscopy determination (55).…”

Section: Topological Organization Of a Tbp-tfiib-tfiif-rnapii-tfiie-tmentioning

confidence: 99%

See 1 more Smart Citation

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

Douziech

Coin

Chipoulet

et al. 2000

Molecular and Cellular Biology

View full text Add to dashboard Cite

The p89/xeroderma pigmentosum complementation group B (XPB) ATPase-helicase of transcription factor IIH (TFIIH) is essential for promoter melting prior to transcription initiation by RNA polymerase II (RNA-PII). By studying the topological organization of the initiation complex using site-specific protein-DNA photo-cross-linking, we have shown that p89/XPB makes promoter contacts both upstream and downstream of the initiation site. The upstream contact, which is in the region where promoter melting occurs (positions ؊9 to ؉2), requires tight DNA wrapping around RNAPII. The addition of hydrolyzable ATP tethers the template strand at positions ؊5 and ؉1 to RNAPII subunits. A mutation in p89/XPB found in a xeroderma pigmentosum patient impairs the ability of TFIIH to associate correctly with the complex and thereby melt promoter DNA. A model for open complex formation is proposed.RNA polymerase II (RNAPII) is the multisubunit enzyme that synthesizes eukaryotic mRNA. The two largest RNAPII subunits, Rpb1 and Rpb2, which are homologous to the ␤Ј and ␤ subunits of prokaryotic RNA polymerases, possess the catalytic activity of the enzyme (5, 71). Rpb1 contains a repeated heptapeptide in its carboxyl-terminal domain (CTD) that becomes highly phosphorylated during the passage from initiation to elongation of transcription (8). Initiation of transcription by RNAPII in vitro requires a set of general transcription initiation factors including TATA-binding protein (TBP), transcription factor IIB (TFIIB), TFIIE, TFIIF, and TFIIH (21,44). TBP binds to the TATA element of promoters and induces an ϳ90°bend in the DNA helix (27,30). TFIIB associates with the TBP-promoter complex (1, 37) and makes promoter contacts on each side of the DNA bend centered on the TATA box (6, 31). TFIIF, which is composed of two subunits called RNAPII-associated proteins 74 and 30 (RAP74 and RAP30), tightly binds to RNAPII and allows the binding of the enzyme to a TBP-TFIIB-promoter complex (4, 16). TFIIE is also composed of two subunits, TFIIE56 and TFIIE34 (25,41), that stabilize the association of RNAPII with the preinitiation complex (50). A complex containing TBP, TFIIB, TFIIE, TFIIF, and RNAPII is capable of initiating transcription on a premelted linear template (23, 45, 61) and on a negatively supercoiled template (46,62,64). Both TFIIE and TFIIF have been shown to play a role in the TFIIH-independent melting of the promoter DNA around the transcription initiation site (TIS) (23,45). TFIIE is also required for the association of TFIIH with the preinitiation complex and the regulation of TFIIH activities (35,39,40). A complex containing TBP, TFIIB, TFIIE, TFIIF, TFIIH, and RNAPII is capable of melting promoter DNA in a region between nucleotides Ϫ9 and ϩ2 of a linear template (22,24,26,69). Open complex formation requires the hydrolysis of the ␤-␥ bond of ATP by the helicaseATPase activity of TFIIH (22,24).Mammalian TFIIH is a nine-subunit complex responsible both for the melting of the template DNA prior to initiation (54, 57) and during prom...

show abstract

Section: Topological Organization Of a Tbp-tfiib-tfiif-rnapii-tfiie-tmentioning

confidence: 77%

Section: Topological Organization Of a Tbp-tfiib-tfiif-rnapii-tfiie-tmentioning

confidence: 99%

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

Douziech

Coin

Chipoulet

et al. 2000

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…DNase I footprinting analysis revealed that addition of a protein fraction containing TFIIE, TFIIF, and TFIIH to promoter-bound complexes that include RNA polymerase II, TBP, and TFIIB results in specific protection of promoter DNA between positions ϩ20 and ϩ30 (32). Results of two-dimensional electron crystallography performed on yeast RNA polymerase II transcription complexes suggest that TFIIE binds to a polymerase domain that contacts downstream DNA (33)(34)(35)(36); because TFIIE and TFIIH bind specifically to one another (37,38), these findings suggest that TFIIH might be similarly positioned. Results of two recent cross-linking studies are also consistent with the idea that TFIIH makes protein-DNA contacts downstream of the RNA polymerase II initiation complex during transcription initiation and, by extension, during promoter escape (11,12).…”

Section: Discussionmentioning

confidence: 99%

TFIIH action in transcription initiation and promoter escape requires distinct regions of downstream promoter DNA

Spangler

Wang

Conaway

et al. 2001

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

View full text Add to dashboard Cite

TFIIH is a multifunctional RNA polymerase II general initiation factor that includes two DNA helicases encoded by the Xeroderma pigmentosum complementation group B (XPB) and D (XPD) genes and a cyclin-dependent protein kinase encoded by the CDK7 gene. Previous studies have shown that the TFIIH XPB DNA helicase plays critical roles not only in transcription initiation, where it catalyzes ATP-dependent formation of the open complex, but also in efficient promoter escape, where it suppresses arrest of very early RNA polymerase II elongation intermediates. In this report, we present evidence that ATP-dependent TFIIH action in transcription initiation and promoter escape requires distinct regions of the DNA template; these regions are well separated from the promoter region unwound by the XPB DNA helicase and extend, respectively, Ϸ23-39 and Ϸ39 -50 bp downstream from the transcriptional start site. Taken together, our findings bring to light a role for promoter DNA in TFIIH action and are consistent with the model that TFIIH translocates along promoter DNA ahead of the RNA polymerase II elongation complex until polymerase has escaped the promoter.T FIIH is a multifunctional RNA polymerase II general initiation factor that includes two DNA helicases encoded by the Xeroderma pigmentosum complementation group B (XPB) and D (XPD) genes, as well as a cyclin-dependent protein kinase encoded by the CDK7 gene (1). Previous studies have shown that the TFIIH XPB DNA helicase functions at multiple steps to promote efficient transcription initiation and promoter escape by RNA polymerase II. The TFIIH XPB DNA helicase catalyzes ATP(dATP)-dependent formation of the open complex before synthesis of the first phosphodiester bond of nascent transcripts (2), and it is required to suppress premature arrest of very early RNA polymerase II elongation intermediates at promoterproximal sites Ϸ10-12 bp downstream of the transcriptional start site before their escape from the promoter (3-5).In a previous study, we identified a requirement in transcription initiation and promoter escape by RNA polymerase II for promoter DNA extending Ϸ23-50 bp downstream from the transcriptional start site (6). In that study, we showed that synthesis of the first phosphodiester bond of nascent transcripts by RNA polymerase II requires promoter DNA extending Ϸ23-39 bp downstream from the transcriptional start site and that efficient promoter escape by the enzyme requires promoter DNA extending Ϸ39-50 bp downstream from the transcriptional start site. That study, however, did not identify which of the general initiation factors require downstream promoter DNA during these stages of transcription.In this report, we present direct biochemical evidence that TFIIH requires downstream promoter DNA for its action in transcription initiation and promoter escape by RNA polymerase II. In addition, we show that TFIIH function in synthesis of the first phosphodiester bond of nascent transcripts and in promoter escape requires distinct regions of DNA downstream of the tra...

show abstract

“…The inclusion of RNase H prevents the formation of long DNA-RNA hybrids and allows pol II to establish a transcription bubble (34). The tailed-template approach was used to obtain the first crystal structures pol II elongation complexes (35,36). The proteins used in this study included four chaperones (ASF1, NAP1, FACT, and SPT6), two ATP-dependent remodeling machines (SWI/SNF and RSC), and pol II.…”

Section: Rsc-dependent Pol II Elongation Is Stimulated By Nucleosomementioning

confidence: 99%

Histone density is maintained during transcription mediated by the chromatin remodeler RSC and histone chaperone NAP1 in vitro

Kuryan

Kim

Tran

et al. 2012

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

View full text Add to dashboard Cite

ATPases and histone chaperones facilitate RNA polymerase II (pol II) elongation on chromatin. In vivo, the coordinated action of these enzymes is necessary to permit pol II passage through a nucleosome while restoring histone density afterward. We have developed a biochemical system recapitulating this basic process. Transcription through a nucleosome in vitro requires the ATPase remodels structure of chromatin (RSC) and the histone chaperone nucleosome assembly protein 1 (NAP1). In the presence of NAP1, RSC generates a hexasome. Despite the propensity of RSC to evict histones, NAP1 reprograms the reaction such that the hexasome is retained on the template during multiple rounds of transcription. This work has implications toward understanding the mechanism of pol II elongation on chromatin. N ucleosomes pose a strong barrier to RNA polymerase II (pol II) elongation (1-3). An understanding of how this barrier is overcome will reveal principles central to all eukaryotic organisms. Genome-wide chromatin immunoprecipitation analyses reveal that histone density is inversely proportional to transcriptional activity (4). However, with the exception of the heat shock loci (5), most genes maintain limited nucleosome density in the coding region during transcription. Maintenance of nucleosome density prevents cryptic transcription, which can have potentially deleterious effects on gene expression and genomic integrity (6).Transcription through chromatin in vivo requires ATP-dependent remodeling machines, histone modification enzymes, and histone chaperones (7). ATP-dependent remodeling enzymes such as SWI/SNF and remodels structure of chromatin (RSC) can mobilize and/or evict nucleosomes to create an unimpeded DNA template for transcription (8). These enzymes are found at the promoters of genes and within ORFs (9-11). Furthermore, SWI/SNF has been shown to travel with pol II in vivo, evicting histones on active genes (12), and RSC has been shown to directly interact with the RNA pol subunit Rpb5 (13). In mammalian cells, pol II pauses at an artificially introduced 601 positioning sequence when SWI/ SNF is knocked down by RNAi (14). Histone chaperones such as ASF1, SPT6, and FACT (SPT16/POB3) also travel with pol II throughout transcription and probably assist in the removal of histones in front of pol II and redeposition behind (15-17). Indeed, one study suggests that FACT redeposits the original histones behind pol II (18).Studies of pol II elongation on chromatin in vitro have revealed insights into the mechanism. Experiments by Studitsky and coworkers showed that pol II frequently stalls and backtracks from the nucleosome (19). Higher ionic strength, which weakens DNA-histone contacts, abrogates the barrier allowing pol II to pass (20). The reaction is stimulated by TFIIS (19). Although nucleosomal passage by pol II occurs at physiological salt concentration (i.e., 150 mM), the efficiency increases with increasing ionic strength (20). Further, one orientation of the 601 positioned nucleosome is more permissive to tran...

show abstract

Electron Crystal Structure of an RNA Polymerase II Transcription Elongation Complex

Cited by 77 publications

References 41 publications

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

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

TFIIH action in transcription initiation and promoter escape requires distinct regions of downstream promoter DNA

Histone density is maintained during transcription mediated by the chromatin remodeler RSC and histone chaperone NAP1 in vitro

Contact Info

Product

Resources

About