A Unified Polymerase Mechanism for Nonhomologous DNA and RNA Polymerases

Steitz, T.A.; Smerdon, Stephen J.; Jäger, Joachim; Cm, Joyce

doi:10.1126/science.7528445

Cited by 275 publications

(215 citation statements)

References 23 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: 88%

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

Sainsbury

Niesser

Cramer

2012

Nature

174

192

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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: 88%

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

Sainsbury

Niesser

Cramer

2012

Nature

174

192

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“…The first detailed structure-based information about the mechanism of DNA polymerases emerged from structural studies (Steitz et al 1994), which discovered the so-called ' two metal mechanism '. A typical polymerase active site is described in Fig.…”

Section: Dna Polymerases and Replication Fidelity 431 Introductionmentioning

confidence: 99%

“…39, where the precise reaction pathway and the contributions to the catalytic effect are not at all obvious from the crystal structure. The original structural studies were unable to resolve the question of whether the proton on the primer 3k hydroxyl group goes to the carboxylate of either Asp 256 or Glu 357 (the authors proposed that there is no need for the participation of an amino acid sidechain for nucleophile activation in the chemical step), or what the precise role of the Mg 2+ cations in the active site is (see also the controversy in Steitz et al 1994). In fact, it was suggested by (Steitz et al 1994) that Mg 2+ (b) helps the reaction by steric effects, following Westheimer's idea (see discussion in Fothergill et al 1995), but, the first theoretical study of this system (Fothergill et al 1995) established that the effect of this magnesium cation is entirely electrostatic.…”

Section: Dna Polymerases and Replication Fidelity 431 Introductionmentioning

confidence: 99%

“…The original structural studies were unable to resolve the question of whether the proton on the primer 3k hydroxyl group goes to the carboxylate of either Asp 256 or Glu 357 (the authors proposed that there is no need for the participation of an amino acid sidechain for nucleophile activation in the chemical step), or what the precise role of the Mg 2+ cations in the active site is (see also the controversy in Steitz et al 1994). In fact, it was suggested by (Steitz et al 1994) that Mg 2+ (b) helps the reaction by steric effects, following Westheimer's idea (see discussion in Fothergill et al 1995), but, the first theoretical study of this system (Fothergill et al 1995) established that the effect of this magnesium cation is entirely electrostatic. Similarly, the theoretical study further clarified the problems with the idea that the carboxylate groups of the negatively charged amino acids are stabilizing the attacking hydroxyl group, pointing out that, in fact, the role of these negatively charged residues is most likely simply to position and shield the positive charge on the cationic Mg 2+ , which will in turn make it much harder for the Mg 2+ to lower the pK a of the hydroxide, and therefore, that the most likely base is simply just another OH x anion from the bulk.…”

Section: Dna Polymerases and Replication Fidelity 431 Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Why nature really chose phosphate

Kamerlin

Sharma

Prasad

et al. 2013

Quart. Rev. Biophys.

316

432

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Phosphoryl transfer plays key roles in signaling, energy transduction, protein synthesis, and maintaining the integrity of the genetic material. On the surface, it would appear to be a simple nucleophile displacement reaction. However, this simplicity is deceptive, as, even in aqueous solution, the low-lying d-orbitals on the phosphorus atom allow for eight distinct mechanistic possibilities, before even introducing the complexities of the enzyme catalyzed reactions. To further complicate matters, while powerful, traditional experimental techniques such as the use of linear free-energy relationships (LFER) or measuring isotope effects cannot make unique distinctions between different potential mechanisms. A quarter of a century has passed since Westheimer wrote his seminal review, ‘Why Nature Chose Phosphate’ (Science 235 (1987), 1173), and a lot has changed in the field since then. The present review revisits this biologically crucial issue, exploring both relevant enzymatic systems as well as the corresponding chemistry in aqueous solution, and demonstrating that the only way key questions in this field are likely to be resolved is through careful theoretical studies (which of course should be able to reproduce all relevant experimental data). Finally, we demonstrate that the reason that naturereallychose phosphate is due to interplay between two counteracting effects: on the one hand, phosphates are negatively charged and the resulting charge-charge repulsion with the attacking nucleophile contributes to the very high barrier for hydrolysis, making phosphate esters among the most inert compounds known. However, biology is not only about reducing the barrier to unfavorable chemical reactions. That is, the same charge-charge repulsion that makes phosphate ester hydrolysis so unfavorable also makes it possible to regulate, by exploiting the electrostatics. This means that phosphate ester hydrolysis can not only be turned on, but also be turned off, by fine tuning the electrostatic environment and the present review demonstrates numerous examples where this is the case. Without this capacity for regulation, it would be impossible to have for instance a signaling or metabolic cascade, where the action of each participant is determined by the fine-tuned activity of the previous piece in the production line. This makes phosphate esters the ideal compounds to facilitate life as we know it.

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“…We depict in Fig. 3, the peptide conformation bound to the active site, which is seen to be ideally poised for a two-metalion catalyzed phosphoryl transfer from ATP to the bound tyrosine: 44,64 i.e., the relative positions of the ATP, Mg 2+ ions, and the catalytic aspartates are in good alignment. Encouraged by this result, we performed peptide docking simulations for two substrate peptides (each being seven amino acids long) corresponding to the Y1068 (VPEYINQ) and Y1173 (NAEYLRV) sites of the C-terminal tail for both wildtype and L834R mutant structures.…”

Section: Constitutive Activation Of the L834r Mutantmentioning

confidence: 99%

A Multiscale Computational Approach to Dissect Early Events in the Erb Family Receptor Mediated Activation, Differential Signaling, and Relevance to Oncogenic Transformations

et al. 2007

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Abstract-We describe a hierarchical multiscale computational approach based on molecular dynamics simulations, free energy-based molecular docking simulations, deterministic network-based kinetic modeling, and hybrid discrete/ continuum stochastic dynamics protocols to study the dimermediated receptor activation characteristics of the Erb family receptors, specifically the epidermal growth factor receptor (EGFR). Through these modeling approaches, we are able to extend the prior modeling of EGF-mediated signal transduction by considering specific EGFR tyrosine kinase (EGFRTK) docking interactions mediated by differential binding and phosphorylation of different C-terminal peptide tyrosines on the RTK tail. By modeling signal flows through branching pathways of the EGFRTK resolved on a molecular basis, we are able to transcribe the effects of molecular alterations in the receptor (e.g., mutant forms of the receptor) to differing kinetic behavior and downstream signaling response. Our molecular dynamics simulations show that the drug sensitizing mutation (L834R) of EGFR stabilizes the active conformation to make the system constitutively active. Docking simulations show preferential characteristics (for wildtype vs. mutant receptors) in inhibitor binding as well as preferential enhancement of phosphorylation of particular substrate tyrosines over others. We find that in comparison to the wildtype system, the L834R mutant RTK preferentially binds the inhibitor erlotinib, as well as preferentially phosphorylates the substrate tyrosine Y1068 but not Y1173. We predict that these molecular level changes result in preferential activation of the Akt signaling pathway in comparison to the Erk signaling pathway for cells with normal EGFR expression. For cells with EGFR over expression, the mutant over activates both Erk and Akt pathways, in comparison to wildtype. These results are consistent with qualitative experimental measurements reported in the literature. We discuss these consequences in light of how the network topology and signaling characteristics of altered (mutant) cell lines are shaped differently in relationship to native cell lines.

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A Unified Polymerase Mechanism for Nonhomologous DNA and RNA Polymerases

Cited by 275 publications

References 23 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

Why nature really chose phosphate

A Multiscale Computational Approach to Dissect Early Events in the Erb Family Receptor Mediated Activation, Differential Signaling, and Relevance to Oncogenic Transformations

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