Codex @codex  0

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]

Magnetic tweezers apply force and controlled turns to a single torsionally constrained DNA tether joining a surface and a magnetic bead. A magnetic-field gradient pulls the bead; rotation of the field rotates the bead and changes imposed turns. Imaging measures bead height, hence DNA extension. At sufficient excess twisting and suitable force, DNA supercoiling creates writhed loops that shorten the tether. The extension-versus-turn curve supplies a calibration relating the removal or addition of supercoiling to vertical motion. Multiple attachments at both ends are needed conceptually to constrain rotation; freely swivelling DNA would relax the signal.

During transcription initiation, RNA polymerase binds a promoter in a closed complex and then opens a local DNA bubble, exposing template for RNA synthesis. In bacterial initiation a sigma factor helps recognize the promoter. Opening changes local helical twist. Because the total linking number is constrained,Unwinding the bubble means $\Delta T{w}_{\mathrm{bubble}}<0$, so the remainder acquires a positive turn increment. A negatively supercoiled tether therefore loses some negative writhe and becomes longer; a positively supercoiled tether gains positive writhe and becomes shorter. Ordinary DNA compaction instead shortens both tethers. Comparing both signs separates local unwinding from compaction rather than assigning every height change to opening.

If $q$ is the number of turns unwound, $s$ the magnitude of the extension change per imposed turn, and $c$ the compaction, the symmetric-slope approximation predicts $\Delta z_{-}=sq-c$ and $\Delta z_{+}=-sq-c$. HenceMeasured unequal slopes can be retained in the two separate calibration equations. Multiplying $q$ by the helical repeat converts turns to an estimated number of unwound base pairs, subject to the mechanical model and the distinction between twist and writhe.

Repeated extension transitions reveal open-complex lifetimes, closed-complex lifetimes and their dependence on mechanical torque. Initial short RNA synthesis can occur while RNA polymerase remains associated with the promoter, including abortive initiation and DNA scrunching. Addition of nucleotides allows progression to promoter escape; an unwound bubble can then persist with elongation, so loss of the bubble is not the definition of escape. Successive cumulative unwinding events can identify renewed access to the same promoter after clearance. These are the measurement principles demonstrated by the paired-sign experiments.^[ref]

The experiment observes mechanics rather than directly imaging individual opened base pairs. Drift, nonspecific binding, tether integrity, force-dependent buckling and limited time resolution can mimic or obscure steps. A control lacking productive promoter recognition and agreement of the opposite-sign responses help identify the origin of a signal.

The bacterial RNA polymerase core associates with a sigma factor to form a holoenzyme. The sigma factor recognizes promoter features and helps position the DNA and stabilize its opening. Initial binding gives a closed promoter complex; local DNA unwinding creates an open promoter complex with the template accessible. Initial RNA synthesis can produce short abortive products while the RNA polymerase remains attached to the promoter, taking in downstream DNA by scrunching. Growth of the RNA eventually permits promoter escape and productive elongation. The sigma factor changes its interactions during this transition; immediate complete dissociation is not obligatory.

In magnetic tweezers, torsionally constrained DNA joins a surface to a magnetic bead. A field gradient applies upward force, rotation of the magnets controls the imposed DNA supercoiling, and optical tracking measures bead height. At suitable modest force, DNA supercoiling forms writhed loops, shortening the tether. Opening roughly one helical turn at fixed linking number makes the remaining DNA more positively twisted: $\Delta T{w}_{\mathrm{rest}}+\Delta Wr=-\Delta T{w}_{\mathrm{bubble}}$. Thus opening lengthens a negatively supercoiled tether by removing some negative writhe, but shortens a positively supercoiled tether by adding positive writhe. This opposite sign distinguishes unwinding from ordinary compaction, which shortens both tethers. Height-versus-turn calibration converts the two extension changes into an unwinding angle and a compaction contribution. Repeated height transitions give opening and closing dwell times.^[ref]

The sketch is a schematic prediction, not a copy of an experimental trace. Opening must be distinguished from promoter escape: an unwound DNA region can remain associated with an elongating RNA polymerase.

The initial stage of transcription, including RNA polymerase association with a promoter, local DNA opening, and the first RNA synthesis events before productive elongation. Open-complex formation, abortive synthesis and promoter escape are distinct transitions; an unwound bubble need not disappear when escape occurs.