Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution

Gnatt, Averell; Cramer, Patrick; Fu, Tsu-Ju; Bushnell, David; Kornberg, Roger D.

doi:10.1126/science.1059495

Cited by 850 publications

(1,016 citation statements)

References 41 publications

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“…In contrast, the downstream DNA in the Pol I PIC is in a similar position to previous DNA‐bound structures of Pol I (Neyer et al , 2016; Tafur et al , 2016) and Pol II (Gnatt et al , 2001; Fig 2C) demonstrating that downstream DNA is retained inside the DNA binding cleft during the transition from initiation to elongation. These findings also agree with previous structures showing that Pol I and Pol II can position downstream DNA correctly in the absence of transcription factors and RNA (Cheung et al , 2011; Tafur et al , 2016).…”

Section: Resultssupporting

confidence: 78%

Structural insights into transcription initiation by yeast RNA polymerase I

et al. 2017

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In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre‐rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi‐subunit pre‐initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo‐EM structure of the 18‐subunit yeast Pol I PIC bound to a transcription scaffold. The cryo‐EM map reveals an unexpected arrangement of the DNA and CF subunits relative to Pol I. The upstream DNA is positioned differently than in any previous structures of the Pol II PIC. Furthermore, the TFIIB‐related subunit Rrn7 also occupies a different location compared to the Pol II PIC although it uses similar interfaces as TFIIB to contact DNA. Our results show that although general features of eukaryotic transcription initiation are conserved, Pol I and Pol II use them differently in their respective transcription initiation complexes.

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Section: Resultssupporting

confidence: 78%

Structural insights into transcription initiation by yeast RNA polymerase I

et al. 2017

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“…Template bases in positions +1/+2 are twisted against each other by about 901 in structures of the undamaged elongation complex 9,13 , but such twisting is impossible for nucleotides that are covalently linked in dinucleotide lesions, giving rise to a translocation barrier 8 . To determine whether translocation of the cisplatin lesion is indeed impaired, we crystallized complex B, which was designed to contain the dimer at positions +1/+2 (scaffold B, Fig.…”

Section: Impaired Entry Of Lesions Into the Active Sitementioning

confidence: 99%

Mechanism of transcriptional stalling at cisplatin-damaged DNA

Damsma

Alt

Brueckner

et al. 2007

Nat Struct Mol Biol

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The anticancer drug cisplatin forms 1,2-d(GpG) DNA intrastrand cross-links (cisplatin lesions) that stall RNA polymerase II (Pol II) and trigger transcription-coupled DNA repair. Here we present a structure-function analysis of Pol II stalling at a cisplatin lesion in the DNA template. Pol II stalling results from a translocation barrier that prevents delivery of the lesion to the active site. AMP misincorporation occurs at the barrier and also at an abasic site, suggesting that it arises from nontemplated synthesis according to an 'A-rule' known for DNA polymerases. Pol II can bypass a cisplatin lesion that is artificially placed beyond the translocation barrier, even in the presence of a G . A mismatch. Thus, the barrier prevents transcriptional mutagenesis. The stalling mechanism differs from that of Pol II stalling at a photolesion, which involves delivery of the lesion to the active site and lesion-templated misincorporation that blocks transcription.Cisplatin (cis-diamminedichloroplatinum(II)) is a widely used anticancer drug that forms DNA adducts that interfere with replication and transcription 1 . The most frequent cisplatin DNA adducts are 1,2-d(GpG) intrastrand cross-links, or cisplatin lesions, in which platinum coordinates the N7 atoms of adjacent guanosines in a DNA strand 2 (Fig. 1a). A cisplatin lesion in the DNA template strand blocks transcription elongation by the single-subunit RNA polymerase from phage T7 (ref.3) and by Pol II 4-6 , and leads to stable polymerase stalling 7 . The stalled Pol II elongation complex can be bound by the elongation factor TFIIS, which stimulates polymerase back-tracking and 3¢ RNA cleavage 6 . A small fraction of polymerases can apparently read through a cisplatin lesion 6 .The detailed molecular mechanisms of cisplatin DNA adduct processing by nucleic acid polymerases are not understood. Here we have used a combination of X-ray crystallography and RNA-extension assays to derive the molecular mechanism of Saccharomyces cerevisiae Pol II stalling at a cisplatin lesion. Comparison of the results with our previous analysis of Pol II stalling at a DNA photolesion, a TT cyclobutane pyrimidine dimer 8 (CPD, Fig. 1a), reveals that the two types of dinucleotide lesions trigger transcriptional stalling by different mechanisms. RESULTS Structure of a cisplatin-damaged Pol II elongation complexTo elucidate recognition of cisplatin-induced DNA damage by transcribing Pol II, we carried out a structure-function analysis of elongation complexes containing a cisplatin lesion in the template DNA strand. Elongation complexes were reconstituted from the 12-subunit S. cerevisiae Pol II and nucleic acid scaffolds as described 8,9 .A cisplatin lesion was first incorporated at registers +2/+3 of the template strand, directly downstream of the NTP-binding site at register +1 (scaffold A, Fig. 1b). The crystal structure of the resulting cisplatin-damaged elongation complex (complex A) was determined at 3.8-Å resolution (Fig. 1c,d and Methods). A very strong peak in the anomalous differe...

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“…This structure revealed that the DNA-RNA hybrid is present in the enzyme cleft, and provided important insights into elongation [9]. Experimental limitations of this system have been overcome in recent studies by reconstituting ECs with synthetic nucleic acids.…”

Section: Rna Polymerase II Elongation Complex Structuresmentioning

confidence: 97%

“…Although an atomic model of the 10-subunit core of Pol II was available [8,9], only backbone models of complete 12-subunit Pol II, which comprises the core and the Rpb4/7 subcomplex, had been reported [10,11]. A new structure of free Pol II subcomplex Rpb4/7 has now enabled refinement of an atomic model of complete Pol II [12 ].…”

Section: Introductionmentioning

confidence: 99%

The dynamic machinery of mRNA elongation

Armache

Kettenberger

Cramer

2005

Current Opinion in Structural Biology

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Two complementary X-ray studies of the interaction between RNA polymerase II and nucleic acids have improved our understanding of mRNA elongation. These studies suggest how RNA polymerase II unwinds DNA, how it separates the RNA product from the DNA template and how it incorporates nucleoside triphosphate (NTP) substrates into the growing RNA chain. The tunable polymerase active center apparently allows repositioning of a catalytic metal ion, rotation of NTPs before their incorporation, RNA repositioning by a transcript cleavage factor, and modulation of enzyme activity by a bacterial small molecule regulator and its protein cofactor.

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Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution

Cited by 850 publications

References 41 publications

Structural insights into transcription initiation by yeast RNA polymerase I

Structural insights into transcription initiation by yeast RNA polymerase I

Mechanism of transcriptional stalling at cisplatin-damaged DNA

The dynamic machinery of mRNA elongation

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