Dataset Information

Binding of Mn-deoxyribonucleoside triphosphates to the active site of the DNA polymerase of bacteriophage T7.

ABSTRACT: Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg2+, as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg2+ to an active site because Mg2+ is spectroscopically silent and Mg2+ binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg2+ with Mn2+:Mn2+ that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn2+ is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn2+ that is free in solution and Mn2+ bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.

SUBMITTER: Akabayov B

PROVIDER: S-EPMC3676745 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Binding of Mn-deoxyribonucleoside triphosphates to the active site of the DNA polymerase of bacteriophage T7.

Akabayov Barak B Richardson Charles C CC

Powder diffraction 20110601 2

Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg2+, as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg2+ to an active site because Mg2+ is spectroscopically silent and Mg2+ binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg2+ w ...[more]

PMID: 23761703

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Project description:RNA polymerase (RNAP) is the primary machine responsible for transcription. Its ability to distinguish between correct (cognate) and incorrect (noncognate) nucleoside triphosphates (NTPs) is important for fidelity control in transcription. In this work, we investigated the substrate selection mechanism of T7 RNAP from the perspective of energetics. The dissociation free energies were determined for matched and unmatched base pairs in the preinsertion complex using the umbrella sampling method. A clear hydrogen-bond-rupture peak is observed in the potential of mean force curve for a matched base pair, whereas no such peaks are present in the position of mean force profiles for unmatched ones. The free-energy barrier could prevent correct substrates from being separated from the active site. Therefore, when NTPs diffuse into the active site, correct ones will stay for chemistry once they establish effective base pairing contacts with the template nucleotide, whereas incorrect ones will be withdrawn from the active site and rejected back to solution. This result provides an important energy evidence for the substrate selection mechanism of RNAP. Then we elucidated energetics and molecular details for correct NTP binding to the active site of the insertion complex. Our observations reveal that strong interactions act on the triphosphate of NTP to constrain its movement, whereas relatively weak interactions serve to position the base in the correct conformation. Triple interactions, hydrophobic contacts from residues M635 and Y639, base stacking from the 3' RNA terminal nucleotide, and base pairing from the template nucleotide act together to position the NTP base in a catalytically competent conformation. At last, we observed that incorrect NTPs cannot be as well-stabilized as the correct one in the active site when they are misincorporated in the insertion site. It is expected that our work can be helpful for comprehensively understanding details of this basic step in genetic transcription.

Dataset Information

Binding of Mn-deoxyribonucleoside triphosphates to the active site of the DNA polymerase of bacteriophage T7.

Publications

Binding of Mn-deoxyribonucleoside triphosphates to the active site of the DNA polymerase of bacteriophage T7.

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