Structural Basis of Transcription: Mismatch-Specific Fidelity Mechanisms and Paused RNA Polymerase II with Frayed RNA

Sydow, Jasmin F.; Brueckner, Florian; Cheung, Alan C. M.; Damsma, Gerke E.; Dengl, Stefan; Lehmann, Elisabeth; Vassylyev, Dmitry G.; Cramer, Patrick

doi:10.1016/j.molcel.2009.06.002

Cited by 171 publications

(212 citation statements)

References 76 publications

(97 reference statements)

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“…In particular, the inferred misincorporation events involving GTP are less frequent, or less able to trigger a long pause, or both. This finding is consistent with a mismatch-specific proofreading mechanism proposed previously (33,36,37), and may be attributable to specific interactions between the TL and the misincorporated 3′ end of the RNA, which could trigger long pauses.…”

Section: Pausing Kinetics Imply a Role For The Trigger Loop In Transcsupporting

confidence: 92%

“…S3). WT RNAPII entered these long pauses approximately once every 10 kb at subsaturating NTP concentrations, a rate that corresponds closely to the frequency for the incorporation of an incorrect nucleotide substrate, as measured by ensemble assays in vitro (36). It has been shown previously that E1103G promotes NTP misincorporation (6,7).…”

Section: Pausing Kinetics Imply a Role For The Trigger Loop In Transcsupporting

confidence: 62%

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Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II

Larson

Zhou

Kaplan

et al. 2012

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

130

187

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During transcription, RNA polymerase II (RNAPII) must select the correct nucleotide, catalyze its addition to the growing RNA transcript, and move stepwise along the DNA until a gene is fully transcribed. In all kingdoms of life, transcription must be finely tuned to ensure an appropriate balance between fidelity and speed. Here, we used an optical-trapping assay with high spatiotemporal resolution to probe directly the motion of individual RNAPII molecules as they pass through each of the enzymatic steps of transcript elongation. We report direct evidence that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, serves as a master regulator of transcription, affecting each of the three main phases of elongation, namely: substrate selection, translocation, and catalysis. Global fits to the force-velocity relationships of RNAPII and its trigger loop mutants support a Brownian ratchet model for elongation, where the incoming NTP is able to bind in either the pre-or posttranslocated state, and movement between these two states is governed by the trigger loop. Comparison of the kinetics of pausing by WT and mutant RNAPII under conditions that promote base misincorporation indicate that the trigger loop governs fidelity in substrate selection and mismatch recognition, and thereby controls aspects of both transcriptional accuracy and rate.optical trap | optical tweezers | Pol II T he transcription of genetic information stably encoded in DNA into a transient RNA message occurs with remarkable fidelity. RNA polymerase (RNAP) incorporates nucleotides into nascent RNA chains at rates of 10-70 s −1 (1, 2), but only inserts the wrong nucleotide approximately once per 100,000 bases, on average (3). Although the energetics of base-pairing is fundamental to transcription fidelity, the discrimination for correctly matched nucleotide substrates is kinetically controlled, and accomplished by active site conformational changes centered on the trigger loop (TL) (4-7). The TL is an evolutionarily conserved mobile element that stabilizes substrate NTPs in the active site of a variety of multisubunit RNA polymerases, including eukaryotic RNAP I, II, and III, as well as bacterial and archaeal RNAP (8). In the case of RNAPII, alteration of the TL has important consequences for activity and fidelity. TL residue His1085, for example, interacts with the bound NTP substrate, and is fully conserved among multisubunit RNA polymerases. The importance of this residue is underscored by the lethality of the H1085A substitution in yeast (7) and catalytic defects resulting from substitutions at this position for both the yeast and bacterial enzymes (7, 9-11). Additionally, a substitution for residue Glu1103 of the TL has been found not only to promote nucleotide misincorporation (6, 7), but also to increase the elongation rate (6,7,12), properties that are jointly consistent with the notion of kinetic proofreading (13). To explore further the interplay between elongation rate and transcriptional fidelity, we determined the effec...

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Section: Pausing Kinetics Imply a Role For The Trigger Loop In Transcsupporting

confidence: 92%

Section: Pausing Kinetics Imply a Role For The Trigger Loop In Transcsupporting

confidence: 62%

Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II

Larson

Zhou

Kaplan

et al. 2012

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

130

187

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“…Endogenous S. cerevisiae 12-subunit Pol II was prepared as described 29 . Full-length TFIIB 11 , TFIIF (S. mikatae Tfg1, S. cerevisiae Tfg2) 4 and TBP 30 (residues 61-240) were prepared as described.…”

Section: Methodsmentioning

confidence: 99%

Conserved architecture of the core RNA polymerase II initiation complex

et al. 2014

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During transcription initiation at promoters of protein-coding genes, RNA polymerase (Pol) II assembles with TBP, TFIIB and TFIIF into a conserved core initiation complex that recruits additional factors. The core complex stabilizes open DNA and initiates RNA synthesis, and it is conserved in the Pol I and Pol III transcription systems. Here, we derive the domain architecture of the yeast core pol II initiation complex during transcription initiation. The yeast complex resembles the human initiation complex and reveals that the TFIIF Tfg2 winged helix domain swings over promoter DNA. An 'arm' and a 'charged helix' in TFIIF function in transcription start site selection and initial RNA synthesis, respectively, and apparently extend into the active centre cleft. Our model provides the basis for further structure-function analysis of the entire transcription initiation complex.

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“…Misincorporations can be corrected with the help of the RNA cleavage stimulatory factor TFIIS (orange) (Kettenberger et al, 2003(Kettenberger et al, , 2004. Proofreading begins with pausing of Pol II, mediated by fraying of the 39-terminal RNA nucleotide (Sydow et al, 2009). DNA lesions such as a cyclobutan pyrimidine dimer (CPD, orange) or a cisplatinum crosslink (orange) can stall transcription and trigger DNA repair Damsma et al, 2007).…”

Section: Transcription Elongation Is Well Understoodmentioning

confidence: 99%

“…The high fidelity of Pol II for the incorporation of the correct nucleotide is required to avoid the production of erroneous mRNA chains. Pol II uses different strategies to recognize and remove misincorporated nucleotides (Sydow et al, 2009). One reason for nucleotide misincorporation into the growing RNA chain can be the presence of DNA lesions within the template strand.…”

Section: Transcription Elongation Is Well Understoodmentioning

confidence: 99%

Towards molecular systems biology of gene transcription and regulation

Cramer

2010

Biological Chemistry

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Ten years after the determination of the RNA polymerase II structure, the basic mechanism of mRNA synthesis during gene transcription is known. In the future, the initiation and regulation of transcription must be studied with a combination of structural biology, biochemistry, functional genomics, and computational methods. In this article, the efforts of our laboratory to move from an integrated structural biology of gene transcription towards molecular systems biology of gene regulation are reviewed.

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Structural Basis of Transcription: Mismatch-Specific Fidelity Mechanisms and Paused RNA Polymerase II with Frayed RNA

Cited by 171 publications

References 76 publications

Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II

Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II

Conserved architecture of the core RNA polymerase II initiation complex

Towards molecular systems biology of gene transcription and regulation

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