BackgroundDuring elongation, multi-subunit RNA polymerases (RNAPs) cycle between phosphodiester bond formation and nucleic acid translocation. In the conformation associated with catalysis, the mobile “trigger loop” of the catalytic subunit closes on the nucleoside triphosphate (NTP) substrate. Closing of the trigger loop is expected to exclude water from the active site, and dehydration may contribute to catalysis and fidelity. In the absence of a NTP substrate in the active site, the trigger loop opens, which may enable translocation. Another notable structural element of the RNAP catalytic center is the “bridge helix” that separates the active site from downstream DNA. The bridge helix may participate in translocation by bending against the RNA/DNA hybrid to induce RNAP forward movement and to vacate the active site for the next NTP loading. The transition between catalytic and translocation conformations of RNAP is not evident from static crystallographic snapshots in which macromolecular motions may be restrained by crystal packing.ResultsAll atom molecular dynamics simulations of Thermus thermophilus (Tt) RNAP reveal flexible hinges, located within the two helices at the base of the trigger loop, and two glycine hinges clustered near the N-terminal end of the bridge helix. As simulation progresses, these hinges adopt distinct conformations in the closed and open trigger loop structures. A number of residues (described as “switch” residues) trade atomic contacts (ion pairs or hydrogen bonds) in response to changes in hinge orientation. In vivo phenotypes and in vitro activities rendered by mutations in the hinge and switch residues in Saccharomyces cerevisiae (Sc) RNAP II support the importance of conformational changes predicted from simulations in catalysis and translocation. During simulation, the elongation complex with an open trigger loop spontaneously translocates forward relative to the elongation complex with a closed trigger loop.ConclusionsSwitching between catalytic and translocating RNAP forms involves closing and opening of the trigger loop and long-range conformational changes in the atomic contacts of amino acid side chains, some located at a considerable distance from the trigger loop and active site. Trigger loop closing appears to support chemistry and the fidelity of RNA synthesis. Trigger loop opening and limited bridge helix bending appears to promote forward nucleic acid translocation.

Cytochrome c oxidase (CcO) converts the energy from redox and oxygen chemistry to support proton translocation and create a transmembrane DeltamuH(+) used for ATP production. Molecular dynamics (MD) simulations were carried out to probe for the formation water chains capable of participating in proton translocation. Attention was focused on the region between and above the a and a(3) hemes where well-defined water chains have not been identified in crystallographic studies. An arginine (R481) (Rhodobacter sphaeroides numbering), positioned between the D-propionates of the hemes, had been mutated in vivo to lysine and showed to have altered activity consistent with an altered proton conductance [Qian, J., Mills, D. A., Geren, L., Wang, K. F., Hoganson, C. W., Schmidt, B., Hiser, C., Babcock, G. T., Durham, B., Millett, F., and Ferguson-Miller, S. (2004) Role of the conserved arginine pair in proton and electron transfer in cytochrome c oxidase, Biochemistry 43, 5748-5756; also see the accompanying paper by Mills et al.]. This mutant was created in silico, and the MD results for the mutant and wild type were compared to explore the effects on the formation of hydrogen-bonded water chains by this mutation. The simulations reveal the presence of hydrogen-bonded water chains that lead from E286 through the region above the hemes to the Mg(2+), and from E286 to the heme a(3) D-propionate and the binuclear center. The R481K mutant does not form as many, or as extensive, water chains as wild-type CcO, due to a new conformation of residues in a large loop between helices III and IV in subunit I, indicating a reduction in the level of water chain formation in the mutant. This loop appears to play a role in controlling the formation of hydrogen-bonded water chains above the hemes. The results suggest a possible gating mechanism for proton movement that includes key residues W172 and Y175 on the loop and F282 on helix VI.

Various tyrosyl radicals generated by reaction of both native and indomethacin-inhibited ovine prostaglandin H synthase-1 with ethyl hydrogen peroxide were examined by using high-field/high-frequency EPR spectroscopy. The spectra for the initially formed tyrosyl radical commonly referred to as the "wide-doublet" species and the subsequent "wide-singlet" species as well as the indomethacin-inhibited "narrow-singlet" species were recorded at several frequencies and analyzed. For all three species, the g-values were distributed. In the case of the wide doublet, the high-field EPR spectra indicated that dominant hyperfine coupling was likely to be also distributed. The g(x)-values for all three radicals were found to be consistent with a hydrogen-bonded tyrosyl radical. In the case of the wide-doublet species, this finding is consistent with the known position of the radical and the crystallographic structure and is in contradiction with recent ENDOR measurements. The high-field EPR observations are consistent with the model in which the tyrosyl phenyl ring rotates with respect to both the protein backbone and the putative hydrogen bond donor during evolution from the wide-doublet to the wide-singlet species. The high-field spectra also indicated that the g-values of two types of narrow-singlet species, self-inactivated and indomethacin-inhibited, were likely to be different, raising the possibility that the site of the radical is different or that the binding of the inhibitor perturbs the electrostatic environment of the radical. The 130 GHz pulsed EPR experiments performed on the wide-doublet species indicated that the possible interaction between the radical and the oxoferryl heme species was very weak.