Mechanism for De Novo RNA Synthesis and Initiating Nucleotide Specificity by T7 RNA Polymerase

Kennedy, William P.; Momand, Jamila R.; Yin, Yajiang

doi:10.1016/j.jmb.2007.03.041

Cited by 61 publications

(60 citation statements)

References 54 publications

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“…N4 mini-vRNAP had a fourfold higher affinity (50 μM) and a threefold higher k cat (300 min −1 ) for GTP than for GDP (200 μM and 100 min −1 ) at physiological (1 mM) Mg 2þ concentration. As a control, we used T7 RNAP, which does not interact with the 5′ phosphate of the initiating nucleotide (8,26). Accordingly, the kinetic parameters were identical when T7 RNAP initiated with GTP or GDP (200 μM and 20 min −1 ).…”

Section: Resultsmentioning

confidence: 99%

“…The published structure of the T7 RNAP transcript initiation complex (8) poses several problems: (i) Distances between the nucleotide-binding metal and its ligands are significantly greater in this structure (average 4.3 Å) than in the elongation complex (average 2.7 Å) (2); (ii) although Y639 has been shown to discriminate NTP against dNTP at the N site for both transcript initiation and elongation (28), Y639 does not contact the 2′-OH of GTPðþ2Þ in the initiation complex structure; (iii) although the interaction between H784 and the 2-amino group of GTPðþ1Þ was shown to play a role in transcription start site selection (29), the H784 side chain contacts GTPðþ2Þ in the structure; and (iv) no motion of RNAP or of the template DNA strand was observed during substrate loading at the active site (8). Therefore, we suspect that the proposed mechanism of transcript initiation based on this T7 RNAP structure requires reevaluation.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, the X-ray crystal structure of the T7 RNAP initiation complex (8) identified a unique nucleotide-binding site that the authors termed the D site (de novo site), which is distinct from the P site used for transcript elongation. In order to determine whether the N4 vRNAP SCI possesses a D site for GTPðþ1Þ binding, we superposed the N4 SCI with the T7 RNAP initiation (8) and elongation complexes (2) by overlaying their Palm cores including the T/DxxGR motif and motifs A and C (Fig. S6 A and B).…”

Section: Discussionmentioning

confidence: 99%

“…Compared with elongation, the process of initiation has not been well characterized by X-ray crystallography. An X-ray crystal structure of T7 RNAP initiation complex was reported (8), but it was captured by using a substrate analog 3′-deoxyGTP (Fig. S1B).…”

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confidence: 99%

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X-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides

Gleghorn

Davydova

Basu

et al. 2011

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

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We have determined the X-ray crystal structures of the pre-and postcatalytic forms of the initiation complex of bacteriophage N4 RNA polymerase that provide the complete set of atomic images depicting the process of transcript initiation by a single-subunit RNA polymerase. As observed during T7 RNA polymerase transcript elongation, substrate loading for the initiation process also drives a conformational change of the O helix, but only the correct base pairing between the þ2 substrate and DNA base is able to complete the O-helix conformational transition. Substrate binding also facilitates catalytic metal binding that leads to alignment of the reactive groups of substrates for the nucleotidyl transfer reaction. Although all nucleic acid polymerases use two divalent metals for catalysis, they differ in the requirements and the timing of binding of each metal. In the case of bacteriophage RNA polymerase, we propose that catalytic metal binding is the last step before the nucleotidyl transfer reaction. D NA-dependent RNA polymerases (RNAPs) transcribe DNA genetic information into RNA and play a central role in gene expression. RNAP catalyzes a nucleotidyl transfer reaction, which is initiated by the nucleophilic attack of an O3′ oxyanion at the RNA 3′ terminus to the α-phosphate (αP) of the incoming nucleotide, resulting in phosphodiester bond formation and release of pyrophosphate (PPi). Both single-subunit T7 phage-like RNAPs and the multisubunit cellular RNAPs possess two nucleotide-binding sites for loading the RNA 3′ end (P site) and the incoming NTP (N site) (1, 2). A two metal-ion catalytic mechanism has been proposed, as the enzyme possesses two divalent catalytic and nucleotide-binding metal cations chelated by two or three conserved Asp residues (3). The catalytic metal is a Lewis acid, coordinating the RNA 3′-OH lowering its pK a and facilitating the formation of the attacking oxyanion. The nucleotide-binding metal is coordinated by the triphosphate of the incoming nucleotide and stabilizes a pentacovalent phosphate intermediate during the reaction. Both metal ions are proposed to have octahedral coordination at physiological Mg 2þ concentrations (4). During transcript elongation, RNAP carries out the loading of a single nucleotide substrate at the N site followed by a nucleotidyl transfer reaction with the RNA 3′ end at the P site; this cycle is repeated as elongation proceeds. X-ray crystal structures of the single-subunit T7 phage RNAP (2, 5) have depicted the process of transcript elongation in detail and reveal a conformational change of the Fingers subdomain during substrate loading to the active site as also observed in the A family of DNA polymerases (DNAPs) (6, 7).Initiation is the only step in the entire transcription process where two nucleotide substrates are loaded at the active site followed by a nucleotidyl transfer reaction. Compared with elongation, the process of initiation has not been well characterized by X-ray crystallography. An X-ray crystal structure of T7 RNAP initiation complex...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

X-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides

Gleghorn

Davydova

Basu

et al. 2011

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

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“…One of the strategies for trapping elusive intermediate of nucleic acid polymerases is the use of a 3Ј-deoxynucleotide analog (13)(14)(15)18), which lacks the essential O3Ј-nucleophile for the phosphodiester bond formation. The intermediate struc-tures captured by using this nucleotide analog revealed the rotation of Fingers subdomain including the O-helix, which is known as the "closing of Fingers," upon nucleotide binding at the active site and proposed that the DNA rearrangement plus the closing of Fingers is the early fidelity checkpoint of the nucleotide selection.…”

mentioning

confidence: 99%

Watching the Bacteriophage N4 RNA Polymerase Transcription by Time-dependent Soak-trigger-freeze X-ray Crystallography

Basu

Murakami

2013

Journal of Biological Chemistry

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Background:The two-metal-ion mechanism of the nucleotidyl transfer reaction by RNA/DNA polymerases has not been adequately elucidated due to lack of temporal resolution. Results: Soak-trigger-freeze x-ray crystallography revealed structures of natural, time-resolved intermediates of transcript initiation. Conclusion: First structural evidence shows that catalytic metal binds after the nucleotide and nucleotide-binding metal and right before reaction chemistry. Significance: Time-dependent soak-trigger-freeze x-ray crystallography can yield functionally relevant high resolution information to study enzyme reactions.

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Transcription: Initiation

Martin¹,

Mironova²

2008

Wiley Encyclopedia of Chemical Biology

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Initiation of transcription is a complex process, requiring melting of the DNA duplex at a sequence‐defined location, de novo synthesis of an initial dinucleotide, polymerization/growth from that dinucleotide to a larger oligonucleotide in an extended RNA‐DNA hybrid, displacement and threading of the 5′ end of the RNA into an exit channel, and release of the initial promoter contacts. These events are necessarily driven energetically by growth of the RNA‐DNA hybrid. It has long been expected and is now becoming structurally clear that stress inherent in this transformation leads to instability during this phase and is a source of the abortive release of short RNA products that is characteristic of RNA polymerases. That apparently unrelated single and multisubunit RNA polymerases share extensive mechanistic (but not structural) homology, likely reflects the demands of the process.

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Mechanism for De Novo RNA Synthesis and Initiating Nucleotide Specificity by T7 RNA Polymerase

Cited by 61 publications

References 54 publications

X-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides

X-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides

Watching the Bacteriophage N4 RNA Polymerase Transcription by Time-dependent Soak-trigger-freeze X-ray Crystallography

Transcription: Initiation

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