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Incoming links: DNA unwinding measured by magnetic tweezers
Past exam of the biological physics course of the University of Oxford 2015 8 iii Solution 2026-10-25
Single-molecule experiments expose distributions and ordered events that an ensemble average can hide. Several distinct biological systems make this advantage concrete.
- In titin single-molecule force spectroscopy, individual sawtooth drops reveal sequential domain unfolding and contour release. An ensemble stretching curve can smooth away that sequence and obscure variation in unfolding thresholds. Force-dependent waiting-time distributions also distinguish stochastic transitions from a single deterministic breaking strength.
- In a patch clamp recording of one ion channel, discrete open-current levels and closed intervals reveal single-channel conductance, gating dwell times, and ion-channel open probability. An ensemble current alone cannot distinguish a few frequently open channels from many rarely open ones, or reveal a brief intermediate state with the same clarity.
- In Single-molecule observation of F1 rotation, a probe on the motor shaft reports angular progression and pauses associated with the catalytic cycle. Rotation demonstrates mechanical coupling directly; individual stepping and dwell-time statistics can connect chemical turnover to angular motion, whereas bulk ATP consumption alone cannot establish the motor's trajectory.
- In DNA unwinding measured by magnetic tweezers, individual extension changes resolve the waiting time and extent of open promoter complex formation by RNA polymerase. Separate binding, opening, and later movements can otherwise merge into one population-averaged response.
These methods also have limitations: the attached probe or applied load may perturb the molecule, inactive or heterogeneous molecules may be overrepresented, and finite temporal resolution can hide fast steps. Repeated independent trajectories and appropriate controls are needed to connect a single observed event to a reproducible mechanism. The gain is access to trajectories and heterogeneity, rather than an assumption that one molecule alone represents every molecule.
At initiation, RNA polymerase recognizes a promoter and forms a closed bound complex; bacterial promoter recognition involves its specificity factor. Local strand opening exposes the template in an open complex, after which initial RNA synthesis can precede promoter escape into elongation. Binding, opening and productive escape are distinct transitions.
For DNA unwinding measured by magnetic tweezers, magnetic tweezers report the mechanics of a torsionally constrained DNA tether through the position of an attached magnetic bead. Axial magnetic force extends the tether, while imposed rotation controls its linking constraint. Video localization measures extension changes. The important physical relation isOpening a small helical region decreases twist, so the rest of the tether acquires compensating positive writhe. On a negatively supercoiled tether this removes some writhe magnitude and raises its extension; on a positively supercoiled tether it increases writhe magnitude and lowers extension. A change with opposite signs under the two torsional conditions therefore distinguishes unwinding from simple compaction, which would shorten both tethers. Calibrating extension against imposed turns converts a mechanical step into a change in helical winding. This is a conceptual readout of local opening, rather than a direct optical image of separated bases.[ref]
Promoter unwinding and promoter clearance by RNA polymerase: Detection by single-molecule DNA nanomanipulation by Andrey Revyakin and others. 2004 study, linking-number argument and opposite extension responses for positive and negative supercoiling
