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RNA polymerase II with open and closed trigger loops: active site dynamics and nucleic acid translocation.

ABSTRACT: RNA polymerase II is the central eukaryotic enzyme in transcription from DNA to RNA. The dynamics of RNA polymerase II is described from molecular-dynamics simulations started from two crystal structures with open and closed trigger loop (TL) forms. Dynamic transitions between neutral and forward translocated states were observed, especially for the downstream DNA duplex. Dynamic rearrangements were also seen in the active site environment, including conformations in which the active site nucleotide assumed a possibly precatalytic conformation in close proximity to the terminal 3'-hydroxyl of the nascent RNA. Because nucleic acid translocation was observed primarily in the simulations with an open TL structure, whereas close approach of the active site nucleotide to the terminal RNA ribose predominantly occurred in the closed TL structure, a modified Brownian ratchet mechanism is proposed whereby thermally driven translocation is only possible with an open TL, and fidelity control and catalysis require TL closing.

SUBMITTER: Feig M

PROVIDER: S-EPMC2956212 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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RNA polymerase II with open and closed trigger loops: active site dynamics and nucleic acid translocation.

Feig Michael M Burton Zachary F ZF

Biophysical journal 20101001 8

RNA polymerase II is the central eukaryotic enzyme in transcription from DNA to RNA. The dynamics of RNA polymerase II is described from molecular-dynamics simulations started from two crystal structures with open and closed trigger loop (TL) forms. Dynamic transitions between neutral and forward translocated states were observed, especially for the downstream DNA duplex. Dynamic rearrangements were also seen in the active site environment, including conformations in which the active site nucleo ...[more]

PMID: 20959099

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Project description:The eight-residue surface loop, 45-52 (Ser, Ala, Val, Gly, Asn, Ala, Glu, Ser), of the homotetrameric protein streptavidin has a "closed" conformation in the streptavidin-biotin complex, where the corresponding binding affinity is one of the strongest found in nature (ΔG ∼ -18 kcal∕mol). However, in most of the crystal structures of apo (unbound) streptavidin, the loop conformation is "open" and typically exhibits partial disorder and high B-factors. Thus, it is plausible to assume that the loop structure is changed from open to closed upon binding of biotin, and the corresponding difference in free energy, ΔF = F(open) - F(closed) in the unbound protein, should therefore be considered in the total absolute free energy of binding. ΔF (which has generally been neglected) is calculated here using our "hypothetical scanning molecular-dynamics" (HSMD) method. We use a protein model in which only the atoms closest to the loop are considered (the "template") and they are fixed in the x-ray coordinates of the free protein; the x-ray conformation of the closed loop is attached to the same (unbound) template and both systems are capped with the same sphere of TIP3P water. Using the force field of the assisted model building with energy refinement (AMBER), we carry out two separate MD simulations (at temperature T = 300 K), starting from the open and closed conformations, where only the atoms of the loop and water are allowed to move (the template-water and template-loop interactions are considered). The absolute F(open) and F(closed) (of loop + water) are calculated from these trajectories, where the loop and water contributions are obtained by HSMD and a thermodynamic integration (TI) process, respectively. The combined HSMD-TI procedure leads to total (loop + water) ΔF = -27.1 ± 2.0 kcal∕mol, where the entropy TΔS constitutes 34% of ΔF, meaning that the effect of S is significant and should not be ignored. Also, ΔS is positive, in accord with the high flexibility of the open loop observed in crystal structures, while the energy ΔE is unexpectedly negative, thus also adding to the stability of the open loop. The loop and the 250 capped water molecules are the largest system studied thus far, which constitutes a test for the efficiency of HSMD-TI; this efficiency and technical issues related to the implementation of the method are also discussed. Finally, the result for ΔF is a prediction that will be considered in the calculation of the absolute free energy of binding of biotin to streptavidin, which constitutes our next project.

Dataset Information

RNA polymerase II with open and closed trigger loops: active site dynamics and nucleic acid translocation.

Publications

RNA polymerase II with open and closed trigger loops: active site dynamics and nucleic acid translocation.

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