Structural Basis of Transcription: Role of the Trigger Loop in Substrate Specificity and Catalysis

Wang, Dong; Bushnell, David; Westover, Kenneth D.; Kaplan, Craig D.; Kornberg, Roger D.

doi:10.1016/j.cell.2006.11.023

Cited by 438 publications

(1,001 citation statements)

References 57 publications

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“…2b). This was consistent with a two-metal ion catalytic mechanism 17,18 , but was unexpected because free Pol II only binds metal A, whereas metal B was observed previously only when a nucleoside triphosphate (NTP) was present 19,20 . The two metal ions are bridged by a rotated aspartate D481 side chain (Fig.…”

supporting

confidence: 85%

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Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

Sainsbury

Niesser

Cramer

2012

Nature

179

195

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The general transcription factor (TF) IIB is required for RNA polymerase (Pol) II initiation and extends with its B-reader element into the Pol II active centre cleft. Low-resolution structures of the Pol II-TFIIB complex 1,2 indicated how TFIIB functions in DNA recruitment, but they lacked nucleic acids and half of the B-reader, leaving other TFIIB functions 3,4 enigmatic. Here we report crystal structures of the Pol II-TFIIB complex from the yeast Saccharomyces cerevisiae at 3.4 Å resolution and of an initially transcribing complex that additionally contains the DNA template and a 6-nucleotide RNA product. The structures reveal the entire B-reader and proteinnucleic acid interactions, and together with functional data lead to a more complete understanding of transcription initiation. TFIIB partially closes the polymerase cleft to position DNA and assist in its opening. The B-reader does not reach the active site but binds the DNA template strand upstream to assist in the recognition of the initiator sequence and in positioning the transcription start site. TFIIB rearranges active-site residues, induces binding of the catalytic metal ion B, and stimulates initial RNA synthesis allosterically. TFIIB then prevents the emerging DNA-RNA hybrid duplex from tilting, which would impair RNA synthesis. When the RNA grows beyond 6 nucleotides, it is separated from DNA and is directed to its exit tunnel by the B-reader loop. Once the RNA grows to 12-13 nucleotides, it clashes with TFIIB, triggering TFIIB displacement and elongation complex formation. Similar mechanisms may underlie all cellular transcription because all eukaryotic and archaeal RNA polymerases use TFIIB-like factors 5 , and the bacterial initiation factor sigma has TFIIB-like topology 1,2 and contains the loop region 3.2 that resembles the B-reader loop in location, charge and function [6][7][8] . TFIIB and its counterparts may thus account for the two fundamental properties that distinguish RNA from DNA polymerases: primer-independent chain initiation and product separation from the template.Our previous X-ray analysis of the Pol II-TFIIB complex at 4.3 Å resolution provided a partial backbone model of TFIIB 1 . To obtain a complete and atomic structure, we co-crystallized Pol II with a TFIIB variant lacking the mobile amino-terminal tail and carboxy-terminal cyclin fold (Methods, Supplementary Table and Supplementary Fig. 1). The resulting Pol II-TFIIB structure at 3.4 Å resolution provides details of the interactions of the four TFIIB domains with Pol II: the B-ribbon with the dock, the B-core N-terminal cyclin fold with the wall, the B-reader helix with the RNA exit tunnel, and the B-linker helix with the coiled-coil of the clamp (Fig. 1).The structure reveals the entire course of the TFIIB polypeptide chain through the Pol II cleft, including the previously lacking 1 B-reader loop (residues 67-79) and a new 'B-reader strand' (residues 80-83). The B-reader loop does not reach the active site, but instead interacts with the Pol II rudder and fork loop ...

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supporting

confidence: 85%

“…The two metal ions are bridged by a rotated aspartate D481 side chain (Fig. 2b), which is also observed in structures of NTP-containing elongation complexes of Pol II 20 and bacterial polymerase 21 . The rearrangement abolishes a contact between metal A and residue D483, and re-orientates the backbone carbonyl in the neighbouring Rpb2 residue D837 (Fig.…”

supporting

confidence: 56%

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

Sainsbury

Niesser

Cramer

2012

Nature

179

195

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“…Binding of the substrate triphosphate may differ in Pol I because the A12.2 hairpin shields the A135 residues Arg 714 and Arg 957, the counterparts of triphosphate-binding Pol II residues. The A12.2 hairpin apparently interferes with closure of the triphosphate-binding trigger loop 27,28 , but may bind the triphosphate and position metal ion B, as suggested for the TFIIS hairpin during RNA cleavage 14 .…”

Section: Expander and Cleft Expansionmentioning

confidence: 92%

RNA polymerase I structure and transcription regulation

Engel

Sainsbury

Cheung

et al. 2013

Nature

200

305

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Transcription of ribosomal RNA by RNA polymerase (Pol) I initiates ribosome biogenesis and regulates eukaryotic cell growth. The crystal structure of Pol I fromthe yeast Saccharomyces cerevisiae at 2.8A˚ resolution reveals all 14 subunits of the 590-kilodalton enzyme, and shows differences to Pol II. An ‘expander’ element occupies the DNA template site and stabilizes an expanded active centre cleft with an unwound bridge helix. A ‘connector’ element invades the cleft of an adjacent polymerase and stabilizes an inactive polymerase dimer. The connector and expander must detach during Pol I activation to enable transcription initiation and cleft contraction by convergent movement of the polymerase ‘core’ and ‘shelf’ modules. Conversion between an inactive expanded and an active contracted polymerase state may generally underlie transcription. Regulatory factors can modulate the core–shelf interface that includes a ‘composite’ active site for RNA chain initiation, elongation, proofreading and termination

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“…These breakthrough discoveries were decisive for awarding the Nobel Prize for Chemistry to Roger Kornberg in 2006. In the following years, many structures of functional complexes of Pol II were determined (Gnatt et al, 2001;Kettenberger et al, 2004;Wang et al, 2006;Vassylyev et al, 2007;Brueckner and Cramer, 2008) that together with functional data led to the first video of the nucleotide addition cycle during transcription elongation . The video facilitates teaching and can be downloaded from the internet at www.LMB.uni-muenchen.de/cramer/PRmaterials/.…”

Section: Transcription Elongation Is Well Understoodmentioning

confidence: 99%

“…Translocation generates a free NTPbinding site and the cycle is repeated. An important role is played by the so-called trigger loop, which closes over the active center to establish a catalytically competent conformation (see Figure 1; Wang et al, 2006). The NTP binds two catalytic metal ions, A and B.…”

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: Role of the Trigger Loop in Substrate Specificity and Catalysis

Cited by 438 publications

References 57 publications

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

RNA polymerase I structure and transcription regulation

Towards molecular systems biology of gene transcription and regulation

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