Two Distinct Modes of Processive Kinesin Movement in Mixtures of ATP and AMP-PNP

An enzyme is frequently conceived of as having a single functional mechanism. This is particularly true for motor enzymes, where the necessity for tight coupling of mechanical and chemical cycles imposes rigid constraints on the reaction pathway. In mixtures of substrate (ATP) and an inhibitor (adenosine 5′-(β,γ-imido)triphosphate or AMP-PNP), single kinesin molecules move on microtubules in two distinct types of multiple-turnover “runs” that differ in their susceptibility to inhibition. Longer (less susceptible) runs are consistent with movement driven by the alternating-sites mechanism previously proposed for uninhibited kinesin. In contrast, kinesin molecules in shorter runs step with AMP-PNP continuously bound to one of the two active sites of the enzyme. Thus, in this mixture of substrate and inhibitor, kinesin can function as a motor enzyme using either of two distinct mechanisms. In one of these, the enzyme can accomplish high-duty-ratio processive movement without alternating-sites ATP hydrolysis

The Cuᴀ Site in Cytochrome c Oxidase: Its Role in Coupling Electron Transport to Proton Pumping

Cytochrome c oxidase contains a copper-ion electron transfer site, CuA, which has previously been found to be unreactive with externally added reagents under conditions in which the protein remains structurally intact. We have studied the reaction of cytochrome oxidase with sodium p-hydroxymercuribenzoate (pHMB) and found that the reaction proceeds, under appropriate conditions, to give an excellent yield of a particular derivative of the CuA center which has electron paramagnetic resonance and near-infrared absorption spectroscopic properties which are distinctly different from those of the unmodified center. Spectroscopic and chemical characterization of the other metal-ion sites of the enzyme reveals little or no effect of the pHMB modification on the structures of and reactions at those sites. Of particular interest is the observation that the modified enzyme still displays a substantial fraction of the native steady-state activity of electron transfer from ferrocytochrome c to O2. Although the modified copper center retains the ability to receive electrons from the powerful reductant Na2S2O4 and to transfer electrons to O2, it is not significantly reduced when the enzyme is treated with milder (higher potential) reductants such as NADH/phenazine methosulfate or the physiological substrate ferrocytochrome c. CuA exhibits many spectroscopic and chemical properties which make it highly atypical of cuproprotein active sites; the singular nature of this site has prompted speculation about the importance of the structural peculiarities of this metal-ion center in the catalytic cycle of the enzyme. In this work, we demonstrate that the unusual features of this site are not prerequisites for competent catalysis of electron transfer and O2 reduction by the enzyme. These observations suggest that a secondary electron transfer pathway not involving CuA exists between the cytochrome c and oxygen binding sites which can function at a rate at least 20% of the total turnover rate of the native enzyme. Cytochrome c oxidase converts free energy released in respiratory electron transport into a metabolically useful form by contributing to the potential gradient across the mitochondrial inner membrane. Both a process involving electron transfer linked proton pumping and a process involving electron transfer from ferrocytochrome c to O2 contribute to the potential gradient. Taken together with those from other recent studies, the results of the experiments support a model for electron transfer in cytochrome oxidase in which CuA and Fea are parts of separate, parallel electron transport pathways between cytochrome c and the cytochrome oxidase O2 reduction site. This model has important implications for the role of CuA in respiratory energy transduction by cytochrome oxidase. It suggests that CuA is the best candidate among the four metal center sites in cytochrome oxidase to be the site of redox-linked proton pumping. In order to explore more fully the mechanistic aspects of energy transduction in cytochrome oxidase, we propose a complete chemical mechanism for the enzyme's proton pump. The mechanism achieves pumping with chemical reaction steps localized at a single redox site within the enzyme; no indirect coupling through protein conformational changes is required. The proposed mechanism is based on a novel redox-linked transition metal ligand substitution reaction. The use of this reaction leads in a straightforward manner to explicit mechanisms for achieving all of the processes determined by Blair, et al. (D.F. Blair, J. Gelles, and S.I. Chan, Biophys. J., in press) to be needed to accomplish redox-linked proton pumping. These processes include: 1) modulation of the energetics of protonation/deprotonation reactions and modulation of the energetics of redox reactions by the structural state of the pumping site; 2) control of the rates of the pump's redox reactions with its electron transfer partners during the turnover cycle (gating of electrons); and 3) regulation of the rates of the protonation/deprotonation reactions between the pumping site and the aqueous phases on the two sides of the membrane during the reaction cycle (gating of protons). The model is the first proposed for the cytochrome oxidase proton pump which is mechanistically complete and specific enough that a realistic assessment can be made of how well the model pump would function as a redox-linked energy transducer. This assessment is accomplished via analyses of the thermodynamic properties and steady-state kinetics expected of the model. These analyses demonstrate that the behavior of a pump based on the model would be very similar to that observed of cytochrome oxidase both in the mitochondrion and purified preparations. Specifically, calculation of the properties of the model pump at equilibrium demonstrates that the behavior expected of the model pump in an electrochemical titration is the same as that observed for the CuA center: a nearly pH-independent midpoint potential of approximately 290 mV. An analysis of the performance of the pump during steady-state turnover demonstrates that the model pump is expected to function efficiently and with good power output under physiological conditions, and that its properties under conditions of varying load are similar to those observed in experiments on respiring mitochondria and on purified cytochrome oxidase reconstituted into artificial lipid vesicles. Although the analysis presented here concerns only a single model of a redox-linked proton pump, it leads to some important general conclusions regarding the mechanistic features of such pumps. The first is that a workable proton pump mechanism does not require large protein conformational changes. Another conclusion is that a redox-linked proton pump need not display a pH-dependent midpoint potential as has frequently been assumed. A final conclusion is that mechanisms for redox-linked proton pumps that involve transition metal ligand exchange reactions are quite attractive because such reactions readily lend themselves to the linked gating processes necessary for proton pumping. Several of the results of this research form the basis of a new approach to studying the role of CuA in energy transduction by cytochrome oxidase. I describe several continuing experimental programs based on this method, and summarize the goals, experimental design, and progress of these studies. I conclude by considering the physiological significance of the new conceptions about the role of CuA in energy transduction by cytochrome oxidase arising from this research.</p

Operator Sequence Alters Gene Expression Independently of Transcription Factor Occupancy in Bacteria

A canonical quantitative view of transcriptional regulation holds that the only role of operator sequence is to set the probability of transcription factor binding, with operator occupancy determining the level of gene expression. In this work, we test this idea by characterizing repression in vivo and the binding of RNA polymerase in vitro in experiments where operators of various sequences were placed either upstream or downstream from the promoter in Escherichia coli. Surprisingly, we find that operators with a weaker binding affinity can yield higher repression levels than stronger operators. Repressor bound to upstream operators modulates promoter escape, and the magnitude of this modulation is not correlated with the repressor-operator binding affinity. This suggests that operator sequences may modulate transcription by altering the nature of the interaction of the bound transcription factor with the transcriptional machinery, implying a new layer of sequence dependence that must be confronted in the quantitative understanding of gene expression

A General Mechanism for Competitor-induced Dissociation of Molecular Complexes

The kinetic stability of non-covalent macromolecular complexes controls many biological phenomena. Here we find that physical models of complex dissociation predict that competitor molecules will, in general, accelerate the breakdown of isolated bimolecular complexes by occluding rapid rebinding of the two binding partners. This prediction is largely independent of molecular details. We confirm the prediction with single-molecule fluorescence experiments on a well-characterized DNA strand dissociation reaction. Contrary to common assumptions, competitor-induced acceleration of dissociation can occur in biologically relevant competitor concentration ranges and does not necessarily imply ternary association of competitor with the bimolecular complex. Thus, occlusion of complex rebinding may play a significant role in a variety of biomolecular processes. The results also show that single-molecule colocalization experiments can accurately measure dissociation rates despite their limited spatiotemporal resolution

Synergistic assembly of human pre-spliceosomes across introns and exons

Most human genes contain multiple introns, necessitating mechanisms to effectively define exons and ensure their proper connection by spliceosomes. Human spliceosome assembly involves both cross-intron and cross-exon interactions, but how these work together is unclear. We examined in human nuclear extracts dynamic interactions of single pre-mRNA molecules with individual fluorescently tagged spliceosomal subcomplexes to investigate how cross-intron and cross-exon processes jointly promote pre-spliceosome assembly. U1 subcomplex bound to the 5\u27 splice site of an intron acts jointly with U1 bound to the 5\u27 splice site of the next intron to dramatically increase the rate and efficiency by which U2 subcomplex is recruited to the branch site/3\u27 splice site of the upstream intron. The flanking 5\u27 splice sites have greater than additive effects implying distinct mechanisms facilitating U2 recruitment. This synergy of 5\u27 splice sites across introns and exons is likely important in promoting correct and efficient splicing of multi-intron pre-mRNAs

Single-Molecule Studies of Origin Licensing Reveal Mechanisms Ensuring Bidirectional Helicase Loading

Loading of the ring-shaped Mcm2–7 replicative helicase around DNA licenses eukaryotic origins of replication. During loading, Cdc6, Cdt1, and the origin-recognition complex (ORC) assemble two heterohexameric Mcm2–7 complexes into a head-to-head double hexamer that facilitates bidirectional replication initiation. Using multi-wavelength single-molecule fluorescence to monitor the events of helicase loading, we demonstrate that double-hexamer formation is the result of sequential loading of individual Mcm2–7 complexes. Loading of each Mcm2–7 molecule involves the ordered association and dissociation of distinct Cdc6 and Cdt1 proteins. In contrast, one ORC molecule directs loading of both helicases in each double hexamer. Based on single-molecule FRET, arrival of the second Mcm2–7 results in rapid double-hexamer formation that anticipates Cdc6 and Cdt1 release, suggesting that Mcm-Mcm interactions recruit the second helicase. Our findings reveal the complex protein dynamics that coordinate helicase loading and indicate that distinct mechanisms load the oppositely oriented helicases that are central to bidirectional replication initiation.National Institutes of Health (U.S.) (NIH grant GM52339)National Institutes of Health (U.S.) (NIH grant R01 GM81648)G. Harold and Leila Y. Mathers FoundationNational Institutes of Health (U.S.) (NIH Pre-Doctoral Training Grant (GM007287))Howard Hughes Medical Institute (Investigator

Single molecule analysis reveals reversible and irreversible steps during spliceosome activation

The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation