Complete RNA Polymerase II Elongation Complex Structure and Its Interactions with NTP and TFIIS

Kettenberger, Hubert; Armache, Karim‐Jean; Cramer, Patrick

doi:10.1016/j.molcel.2004.11.040

Cited by 393 publications

(584 citation statements)

References 46 publications

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“…The DNA-RNA hybrid occupies the upstream positions -1 to -8. The hybrid structure is essentially identical to our previous structures of the complete Pol II elongation complex 9 and the elongation complex containing a CPD lesion at the polymerase active site 8 . However, the downstream DNA duplex adopts a slightly altered position (Fig.…”

Section: Structure Of a Cisplatin-damaged Pol II Elongation Complexsupporting

confidence: 67%

“…Endogenous ten-subunit S. cerevisiae Pol II core enzyme was purified as described 20 except that the anion-exchange step was omitted. Recombinant TFIIS was prepared as described 9 , and synthetic oligonucleotides were annealed for scaffold assembly as described 8 . We assembled Pol II-nucleic acid scaffold complexes by incubating pure core Pol II with two molar equivalents of nucleic acid scaffold in transcription buffer (20 mM HEPES (pH 7.6), 60 mM (NH 4 ) 2 SO 4 , 8 mM MgSO 4 , 10 mM ZnCl 2 , 10% (v/v) glycerol, 10 mM DTT) at 20 1C for 30 min as described 8 .…”

Section: Methodsmentioning

confidence: 99%

“…The cisplatin lesion-containing scaffolds were cocrystallized and the structures were determined essentially as described 8,9 , with minor changes. Stoichiometric Pol II-scaffold complexes were assembled by incubating core Pol II for 10 min with two molar equivalents of nucleic acid scaffold, then incubating this mixture for 20 min with five molar equivalents of recombinant Rpb4-Rpb7 in Pol II buffer (5 mM HEPES (pH 7.25), 40 mM (NH 4 ) 2 SO 4 , 10 mM ZnCl 2 , 10 mM DTT) at 20 1C.…”

Section: Methodsmentioning

confidence: 99%

“…Elongation complexes were reconstituted from the 12-subunit S. cerevisiae Pol II and nucleic acid scaffolds as described 8,9 .…”

Section: Structure Of a Cisplatin-damaged Pol II Elongation Complexmentioning

confidence: 99%

“…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%

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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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Section: Structure Of a Cisplatin-damaged Pol II Elongation Complexsupporting

confidence: 67%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Elongation complexes were reconstituted from the 12-subunit S. cerevisiae Pol II and nucleic acid scaffolds as described 8,9 .…”

Section: Structure Of a Cisplatin-damaged Pol II Elongation Complexmentioning

confidence: 99%

Section: Impaired Entry Of Lesions Into the Active Sitementioning

confidence: 99%

See 3 more Smart Citations

Mechanism of transcriptional stalling at cisplatin-damaged DNA

Damsma

Alt

Brueckner

et al. 2007

Nat Struct Mol Biol

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160

133

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Evolution of Eukaryotic RNA Polymerases

Drouin

Robert

2010

Encyclopedia of Life Sciences

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Ribonucleic acid (RNA) polymerases are responsible for the transcription of deoxyribonucleic acid (DNA) into RNA. The number of protein subunits making up these enzymes has increased during evolution from 5 in Eubacteria to 12 in the common ancestor of Archaea and eukaryotes. Eukaryotes have three RNA polymerases , each transcribing a particular subset of genes. RNA polymerase I transcribes ribosomal RNA genes, RNA polymerase II transcribes messenger RNA genes and RNA polymerase III transcribes 5S and transfer RNA genes. Although RNA polymerase II is still made up of 12 subunits, the number of subunits has increased to 14 in RNA polymerase I and to 17 in RNA polymerase III . These new subunits originated from the permanent recruitment of pre‐existing general transcription factors. Interestingly, the evolution of the three eukaryotic RNA polymerases has been affected not only by adaptive forces but also by the nonadaptive forces due to the concerted evolution of the genes they transcribe. Key Concepts: The larger numbers of protein subunits found in RNA polymerases I and III, relative to RNA polymerase II, are due to the permanent recruitment of general transcription factors. The three universal eukaryotic RNA polymerases have specific functional differences near their respective active sites. During evolution, the three eukaryotic RNA polymerase have been selected to better perform their specific tasks. The rate of evolution of RNA polymerases is proportional to the amount of homogenisation (concerted evolution) experienced by the genes they transcribe. Nonadaptive processes therefore also affect the rate of evolution of RNA polymerase genes.

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Complete RNA Polymerase II Elongation Complex Structure and Its Interactions with NTP and TFIIS

Cited by 393 publications

References 46 publications

Mechanism of transcriptional stalling at cisplatin-damaged DNA

Mechanism of transcriptional stalling at cisplatin-damaged DNA

Evolution of Eukaryotic RNA Polymerases

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