Architecture of the Human and Yeast General Transcription and DNA Repair Factor TFIIH

Luo, Jie; Cimermančič, Peter; Viswanath, Shruthi; Ebmeier, Christopher C.; Kim, Bong Keun; Dehecq, Marine; Raman, Vishnu; Greenberg, Charles H.; Pellarin, Riccardo; Šali, Andrej; Taatjes, Dylan J.; Hahn, Steven; Ranish, Jeff

doi:10.1016/j.molcel.2015.07.016

Cited by 93 publications

(157 citation statements)

References 61 publications

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“…The resulting model explains why residues K95 and K731 of Rad3 (colored cyan in Fig. 2 C and D) both form cross-links to the core subunits of TFIIH (1,16). Although Rad3 was in close proximity to TFIIE, there was little or no contact between them ( Fig.…”

Section: Resultsmentioning

confidence: 94%

Structure of an RNA polymerase II preinitiation complex

Murakami

Tsai

Kalisman

et al. 2015

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

109

120

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The structure of a 33-protein, 1.5-MDa RNA polymerase II preinitiation complex (PIC) was determined by cryo-EM and image processing at a resolution of 6-11 Å. Atomic structures of over 50% of the mass were fitted into the electron density map in a manner consistent with protein-protein cross-links previously identified by mass spectrometry. The resulting model of the PIC confirmed the main conclusions from previous cryo-EM at lower resolution, including the association of promoter DNA only with general transcription factors and not with the polymerase. Electron density due to DNA was identifiable by the grooves of the double helix and exhibited sharp bends at points downstream of the TATA box, with an important consequence: The DNA at the downstream end coincides with the DNA in a transcribing polymerase. The structure of the PIC is therefore conducive to promoter melting, start-site scanning, and the initiation of transcription.-E, -F, and -H, are required for the initiation of RNA polymerase II (pol II) transcription. The GTFs can be assembled with pol II and promoter DNA in a preinitiation complex (PIC) capable of efficient conversion to a transcribing complex (0.1-0.3 transcripts per template). Cryo-EM of the PIC at a resolution of 15-20 Å revealed a bipartite structure, with a P-lobe containing pol II and a G-lobe containing most of the mass of the GTFs (1). A cylinder of electron density, attributed to promoter DNA, was in contact only with GTFs and not with pol II. This architecture was consistent with the pathway of assembly of the PIC, beginning with a complex of promoter DNA and all but one of the GTFs, to which a complex of pol II and the remaining GTF was added (2).A combination of chemical cross-linking and mass spectrometry was used to locate the subunits of the GTFs in the low-resolution cryo-EM electron density map of the PIC. TFIIB was in proximity to promoter DNA at the upstream end of the pol II active center cleft, whereas Ssl2, a subunit of TFIIH, was in apparent contact with promoter DNA at the downstream end of the cleft. TFIIB bridges between pol II and promoter DNA, whereas Ssl2 is a DNA helicase, responsible for unwinding promoter DNA.The association of promoter DNA with the GTFs and not with pol II may be viewed as a fundamental principle of the PIC. Doublestranded DNA is straight and relatively rigid, whereas DNA would need to bend by about 90°to bind in the pol II active center cleft. The GTFs assemble on the double-stranded DNA, position it above the active center, and unwind the DNA. The resulting singlestranded region is flexible and can bend and bind in the pol II cleft.Cryo-EM of the PIC with state-of-the-art instrumentation has resulted in an electron density map at higher resolution. The improved map has confirmed and extended the previous findings and conclusions. ResultsCryo-EM and 3D Reconstruction. A PIC was formed as described (2) on an 86-bp fragment of HIS4 promoter DNA fragment, with pol II, GTFs (TFIIA, TFIIB, TBP, TFIIE, TFIIF, and holo-TFIIH), TFIIS, and with the ad...

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Section: Resultsmentioning

confidence: 94%

Structure of an RNA polymerase II preinitiation complex

Murakami

Tsai

Kalisman

et al. 2015

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

109

120

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“…For CXMS, TAP-purified editosome complexes were mixed with 2 mM or 5 mM BS3, quenched, digested with trypsin, and peptides were fractionated by strong cation-exchange chromatography and analyzed by MS. Crosslinked peptides were identified by searching the MS data against appropriate databases using pLink (76) and Nexus (48,77). A 5% false-discovery rate was used for both pLink and Nexus searches.…”

Section: Methodsmentioning

confidence: 99%

“…S3). This approach identifies the specific crosslinked amino acid residues and uses distance constraints of the cross-linker to define the residues' proximity, thus revealing potential domain-domain interactions (48). Each ∼20S editosome component, except KREPA4, contains many lysine residues distributed throughout the polypeptides (SI Appendix, Table S3), making editosomes good substrates for this technique.…”

mentioning

confidence: 99%

The Architecture of Trypanosoma brucei editosomes

McDermott

Luo

Carnes

et al. 2016

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

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Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei. Editing is catalyzed by three distinct ∼20S editosomes that have a common set of 12 proteins, but are typified by mutually exclusive RNase III endonucleases with distinct cleavage specificities and unique partner proteins. Previous studies identified a network of protein-protein interactions among a subset of common editosome proteins, but interactions among the endonucleases and their partner proteins, and their interactions with common subunits were not identified. Here, chemical cross-linking and mass spectrometry, comparative structural modeling, and genetic and biochemical analyses were used to define the molecular architecture and subunit organization of purified editosomes. We identified intra-and interprotein cross-links for all editosome subunits that are fully consistent with editosome protein structures and previously identified interactions, which we validated by genetic and biochemical studies. The results were used to create a highly detailed map of editosome protein domain proximities, leading to identification of molecular interactions between subunits, insights into the functions of noncatalytic editosome proteins, and a global understanding of editosome architecture.cross-linking | proteomics | Trypanosoma brucei | RNA editing | editosome T he kinetoplastid Trypanosoma brucei is the causative agent of human African trypanosomiasis, which is transmitted by the tsetse fly and is a health threat to millions of people in subSaharan Africa. Kinetoplastids are named for their distinctive mitochondrial DNA network, known as the kinetoplast DNA, which is composed of interlocked DNA maxicircles and minicircles (1, 2). Several identical maxicircles encode two ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs) for 18 mitochondrial proteins, 12 of which undergo posttranscriptional RNA editing (3-9). RNA editing was first described in kinetoplastids (6,8,9), where it is essential (10) and involves insertion and deletion of uridines (Us) to generate translatable mitochondrial transcripts. The sequence information for editing is specified by ∼60-nucleotide guide RNAs (gRNAs) that are encoded in thousands of heterogeneous minicircles (11)(12)(13)(14)(15)(16)(17). Editing occurs by rounds of coordinated catalytic steps that require a number of different enzymes: cleavage of the mRNA substrate by endonucleases, addition of Us by 3′ terminal uridylytransferase (TUTase) or removal of Us by U-specific 3′ exonuclease (exoUase) at insertion or deletion editing sites, respectively, and rejoining of mRNA fragments by RNA ligases. The enzymes that catalyze RNA editing in T. brucei are in ∼20S editosome complexes that also contain proteins with no known catalytic functions (16,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28).There are three similar versions of ∼20S editosomes that have a common set of 12 proteins (SI Appendix, Fig. S1 and Table S1). However, they are compositionally and functionally distinct in that each...

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“…XPD/p44 is part of the multisubunit TFIIH complex, and DNA lesion search, recognition, and verification as well as removal are all tightly regulated via protein-protein interactions (38). Recent studies by Yang and co-workers (28) have demonstrated stalling of XPD helicase activity by bulky DNA lesions (indicating target recognition) in the context of the (seven subunits) TFIIH core complex.…”

Section: Uvrb and Xpd Helicase Activities Are Activated By Proteinmentioning

confidence: 99%

Conservation and Divergence in Nucleotide Excision Repair Lesion Recognition

Wirth

Gross

Roth

et al. 2016

Journal of Biological Chemistry

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Nucleotide excision repair is an important and highly conserved DNA repair mechanism with an exceptionally large range of chemically and structurally unrelated targets. Lesion verification is believed to be achieved by the helicases UvrB and XPD in the prokaryotic and eukaryotic processes, respectively. Using single molecule atomic force microscopy analyses, we demonstrate that UvrB and XPD are able to load onto DNA and pursue lesion verification in the absence of the initial lesion detection proteins. Interestingly, our studies show different lesion recognition strategies for the two functionally homologous helicases, as apparent from their distinct DNA strand preferences, which can be rationalized from the different structural features and interactions with other nucleotide excision repair protein factors of the two enzymes. Nucleotide excision repair (NER)4 is an important DNA repair mechanism with a large range of chemically and structurally unrelated targets. Examples range from bulky DNA adducts to interstrand DNA cross-links caused by antitumor drugs (1-3). In humans, NER is the only repair system for the removal of UV irradiation-induced photoproducts such as the intrastrand cross-linking cyclobutane-pyrimidine dimers (CPDs), and dysfunctional NER is responsible for severe diseases, including xeroderma pigmentosum (XP) (1-3).The mechanism of NER is highly conserved between organisms. In bacteria, NER involves the proteins UvrA, UvrB, and UvrC. In the current model of prokaryotic NER, a hetero-tetrameric complex of UvrA and UvrB (UvrA 2 B 2 ) scans the DNA for lesions. When a lesion is encountered, initial lesion sensing by a dimer of UvrA is based on detection of DNA distortion. Conformational changes in the UvrA 2 B 2 complex result in an unwinding and opening of dsDNA around the lesion, providing an unpaired (bubble) region likely required by UvrB to thread onto one of the ssDNA strands. The helicase UvrB is believed to verify the presence of a lesion by insertion of a ␤-hairpin via interactions with residues at the base of the hairpin (2). Upon lesion verification by UvrB, UvrA dissociates from the complex, and ATP re-binding by UvrB results in the formation of the lesion-specific UvrB-DNA complex, which recruits the NER endonuclease UvrC (UvrBC complex). UvrC carries out two incisions on either side of the lesion. The 12-13-nucleotide (nt)-long ssDNA stretch (2) containing the lesion can then be removed together with the endonuclease by the helicase UvrD, and the resulting gap is filled and sealed by DNA polymerase I and ligase.Eukaryotic NER encompasses a total of ϳ30 proteins, including the xeroderma pigmentosum group proteins (XPA-XPG). Repair can either be initiated by a stalled RNA polymerase in transcription-coupled NER or via global genome NER. In global genome NER, upon initial detection of short destabilized DNA structures by the CEN2-XPC-HR23B complex, the ATPase/ helicase XPB, which is part of the 10-subunit transcription factor IIH (TFIIH) complex, then directly interacts with XPC (4) and...

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Architecture of the Human and Yeast General Transcription and DNA Repair Factor TFIIH

Cited by 93 publications

References 61 publications

Structure of an RNA polymerase II preinitiation complex

Structure of an RNA polymerase II preinitiation complex

The Architecture of Trypanosoma brucei editosomes

Conservation and Divergence in Nucleotide Excision Repair Lesion Recognition

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