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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.

Treat the target as an absorbing sphere of effective capture radius $r_0$, surrounded by a uniform dilute reservoir of RNA polymerase with number density $C_{\infty }$. The steady diffusion equation, spherical symmetry, and boundary conditions giveBy Fick's laws, the inward capture rate per target is $4\pi r_0^2{D}_A{C}^{\prime }(r_0)=4\pi D_A{r}_0{C}_{\infty }$. Multiplying by the target number density and comparing with the bimolecular rate law therefore yields the Smoluchowski association rateIts units here are ${\mathrm{m}}^3{\mathrm{s}}^{-1}$, since the concentrations count particles rather than moles. The absorbing boundary assumes every encounter is productive; orientation constraints would reduce the effective association rate constant.

The Stokes–Einstein relation, for RNA polymerase radius $a=15\mathrm{nm}$ at an illustrative temperature $T=300\mathrm{K}$, givesNeither cell volume nor the effective $r_0$ is specified, so a unique numerical time is unavailable. A representative Escherichia coli volume is $V=1\mu {\mathrm{m}}^3=10^{-18}{\mathrm{m}}^3$. The $40$-base pair target has a DNA contour extent of about $40(0.34)=13.6\mathrm{nm}$; using this as an order-of-magnitude capture radius gives $k_a\simeq 2.50\times 10^{-19}{\mathrm{m}}^3{\mathrm{s}}^{-1}$. For $M=3000$ independent searchers, the target's approximately constant encounter hazard is $M{k}_a/V$, henceUsing half the target's contour extent for $r_0$ instead gives about $2.7\mathrm{ms}$; the spherical capture model is not exact target geometry. One searcher's mean would be about $4\mathrm{s}$ under the first convention, whereas the first arrival among $3000$ is much faster.

If only a fraction $f_3=0.1$ of each searcher's time is available for three-dimensional diffusion, and nonspecific DNA binding neither scans nor delivers the target, the effective searching population is $f_{3}M$. Thus $\boxed{\tau \simeq {\tau }_3/f_3=10{\tau }_3}$, about $13\mathrm{ms}$ under the stated estimate. This assumes stationarity, well-mixed searchers and no additional target-access route; nonspecific binding alone is sequestration, not facilitated diffusion.

A bacterial-type plastid RNA polymerase with core subunits encoded by the plastid genome. Nuclear-encoded sigma factors and associated components mean that its name does not imply autonomous regulation.

An assembly of RNA polymerase and associated factors at a gene promoter before productive RNA synthesis. Its positioning and opening of template DNA help determine initiation.

The transition from initiation at a promoter to productive elongation by RNA polymerase. Initial short RNA products may be released before escape; retention or loss of a sigma factor is not by itself a universal marker of escape.

A ribozyme is an RNA molecule whose folded structure provides catalysis of a chemical reaction. A protein enzyme builds its active site from an amino acid chain; a ribozyme builds a working active site from RNA, sometimes with metal ions and associated proteins. Base pairing can help recognize a substrate, but recognition alone does not establish catalysis: the RNA must promote chemical conversion. A passive messenger RNA template being read by a ribosome is not thereby a ribozyme. See Cech and Herschlag, Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis, sections 1–3.

The surprise is that an information-bearing polymer can also perform chemistry. Examples include the self-splicing group I intron of Tetrahymena thermophila and the catalytic RNA component of bacterial RNase P, discussed below. The ribosome supplies an everyday example: its peptide bond-forming active site is made of ribosomal RNA, although the complete machine also contains proteins. Nissen and colleagues examined structures of a large ribosome subunit with substrate analogues and found RNA contacting those analogues at the catalytic centre. This supports an RNA active site, not a claim that isolated ribosomal RNA can carry out all of biological translation. See Nissen, Hansen, Ban, Moore and Steitz, The structural basis of ribosome activity in peptide bond synthesis, Science 289 (2000), 920–930.

For the Tetrahymena group I intron, RNA splicing removes the intron and joins its flanking exons. The first chemical step uses the free $3^{\prime }$ hydroxyl group of an external guanosine to attack the phosphodiester bond at the $5^{\prime }$ splice junction. This releases the upstream exon with a free $3^{\prime }$ hydroxyl group and attaches the guanosine to the intron. That newly freed hydroxyl group then attacks the phosphodiester bond at the other splice junction, joining the exons and releasing the linear intron. Each step exchanges one backbone connection for another; it is not simply separation of base pairs. See Cech, Zaug and Grabowski, 1981: the phosphoester-transfer mechanism.

The motivating experiment began with an unexpected control. As Cech recounts, Zaug purified a ribosomal RNA precursor from Tetrahymena using a detergent and phenol extraction, then compared test tubes with and without added cell nucleus extract. Incubation in salts and nucleotides released the same amount of small intron RNA in both cases, detected by gel electrophoresis. Repetition made a missing ingredient or accidental omission less plausible. Activity also survived treatments intended to destroy proteins. Nevertheless, absence of added extract did not by itself exclude a tightly attached, resistant protein, or prove that the small RNA had been newly excised rather than released from an already processed complex. See Thomas R. Cech, 1989 Nobel lecture, pp. 655–659: the unexpected control and alternative explanations.

An independent chemical readout addressed the latter problem. Cech mixed precursor RNA with phosphorus-32-labelled guanosine bearing three phosphate groups (GTP), and used gel electrophoresis followed by exposure to X-ray film to locate the label. Label appeared in the excised intron with magnesium present; it was absent when magnesium was omitted or labelled ATP replaced the labelled guanosine reagent. This experiment is described in Cech, 1989 Nobel lecture, pp. 657–658: labelled-guanosine controls. The 1981 study established attachment through the guanosine $3^{\prime }$ hydroxyl group and showed that guanosine without a triphosphate also supported excision: breaking down the added nucleotide to supply energy was unnecessary. See Cech, Zaug and Grabowski, In vitro splicing of the ribosomal RNA precursor of Tetrahymena, Cell 27 (1981), 487–496: primary abstract. Separately, sequencing and terminal analysis found an extra $5^{\prime }$ guanosine absent from the corresponding DNA sequence. See Zaug and Cech, The intervening sequence excised from the ribosomal RNA precursor of Tetrahymena contains a 5-terminal guanosine residue not encoded by the DNA, Nucleic Acids Research 10 (1982), 2823–2838. Together, incorporation and sequence evidence support a new covalent bond, beyond a change in how RNA associates.

Kruger, Grabowski, Zaug, Sands, Gottschling and Cech's 1982 test removed dependence on a precursor purified from Tetrahymena. They cloned the intron and adjacent exons into a DNA template, made precursor RNA with purified bacterial RNA polymerase under conditions suppressing RNA splicing, and removed proteins by detergent–phenol extraction. Denaturing gel electrophoresis separated precursor species. Purified precursor was then incubated with the guanosine reagent, magnesium and salt; matched samples without this incubation supplied a precursor baseline. Their standard assay used $10\mathrm{mM}$ magnesium chloride, $0.1\mathrm{mM}$ of that guanosine reagent, $100\mathrm{mM}$ ammonium sulfate and a buffer solution at pH $7.5$, for $30$ minutes at temperature $37{}^{\circ }\mathrm{C}$, with specified variations in individual experiments. Gel electrophoresis and radioactive-film detection showed excised intron RNA; sequencing verified its $5^{\prime }$ junction. See Kruger et al., Self-Splicing RNA, Cell 31 (1982), 147–157, figures 3–4 and Experimental Procedures.

Excision alone could leave disconnected exons. To test joining, the authors paired labelled RNA with DNA spanning the joined-exon sequence, removed exposed RNA with enzymes, and separated the protected pieces by denaturing gel electrophoresis. Spliced samples yielded protected RNA long enough to span the junction. See Kruger et al., 1982, figure 6: independent evidence for exon ligation. The experimental logic matters: an intron-sized band identifies a candidate product, while sequence and junction assays test its chemical identity.

These controls support sufficiency of the prepared RNA under the assay conditions. They do not show that every RNA self-splices, or that proteins are dispensable inside a biological cell. Many group I introns use proteins to help attain an active fold. See Cech and Herschlag, Group I Ribozymes, section 6: protein facilitation. Nor does a failed assay necessarily identify an inactive nucleotide sequence: unsuitable ions, pH, or temperature can prevent a working active site from forming.

The distinction between self-processing and repeated use became especially clear with bacterial RNase P. In 1983 Guerrier-Takada, Gardiner, Marsh, Pace and Altman separated the RNA and protein components of RNase P from two bacteria. Purified RNA cleaved precursor transfer RNA under conditions containing $60\mathrm{mM}$ magnesium ions, or $10\mathrm{mM}$ magnesium ions plus $1\mathrm{mM}$ spermidine. The protein components alone had no catalytic activity. At $5$–$10\mathrm{mM}$ magnesium without spermidine, both components were needed. Thus the comparison demonstrated an RNA catalyst while also explaining why protein assistance could matter under other conditions. RNase P produces the mature $5^{\prime }$ end of transfer RNA by removing its leader; it acts on a separate substrate and can be used again. See Guerrier-Takada et al., The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme, Cell 35 (1983), 849–857: primary abstract.

Self-splicing demonstrates RNA chemistry, but one precursor completing one rearrangement is not yet evidence for catalytic turnover: repeated conversion of separate substrates by a regenerated catalyst. The self-splicing precursor changes its own connectivity and incorporates external guanosine. In 1986 Zaug and Cech showed that a shortened Tetrahymena intron RNA could cleave and rejoin separate short RNA substrates in a sequence-dependent reaction. This demonstrated an RNA enzyme acting beyond its own self-processing. See Zaug and Cech, The intervening sequence RNA of Tetrahymena is an enzyme, Science 231 (1986), 470–475: primary abstract.

Read the second figure as a counting argument. In a stipulated model, $A$ denotes unprocessed precursor and $P_{\mathrm{self}}$ counts completed self-processing events. Set $A(0)=R_0$, $P_{\mathrm{self}}(0)=0$ and $dA/d\tau =-A$. The exponential function solution gives $P_{\mathrm{self}}/R_0=1-e^{-\tau }$, never exceeding one. Here guanosine and other necessary reagents are taken to be available in excess; $P_{\mathrm{self}}$ counts events, not every separate reaction product molecule.

For a second stipulated model, let an unchanged ribozyme $R$ perform $R+S⟶R+P$ on a separate substrate $S$. Assume the effective law of mass action $dS/dt=-kRS$, no reverse reaction, no ribozyme loss and no inhibited or long-lived bound states. Set $R=R_0$, $S(0)=5R_0$, $P(0)=0$ and $\tau =k{R}_{0}t$. Then $S/R_0=5e^{-\tau }$ and $P/R_0=5(1-e^{-\tau })$, with $S+P=5R_0$. At $\tau =1$, self-processing gives about $0.632$ completed events per initial RNA, whereas the regenerated ribozyme gives about $3.161$ reaction product molecules per initial ribozyme. More than one substrate conversion per initial ribozyme, with appropriate background and identity controls, is evidence for turnover. Product accumulation below that threshold alone cannot distinguish a reusable catalyst from one-shot chemistry. These models illustrate that distinction; actual enzyme kinetics can include folding delays, binding, saturation and product release.

An RNA polymerase that uses RNA as its template. In some RNA interference pathways it helps generate substrates for small-RNA production.

An RNA polymerase used in transcription is an enzyme that synthesizes RNA from a DNA template. Unlike a replicating DNA polymerase, it can start a new strand without a pre-existing paired strand end; initiation still requires the appropriate DNA signals and associated machinery. See Alberts et al.: RNA polymerase and transcription initiation.

A protein that confers promoter-recognition specificity on a bacterial-type RNA polymerase. Plastid sigma factors can be nuclear encoded and imported.

A T7 expression system uses bacteriophage T7 RNA polymerase to transcribe an insert from a recognized promoter. Regulating the supply or activity of that polymerase can control expression. High transcript production can still yield insoluble or inactive protein.

A transcription factor that increases transcription of a target gene. It can act by recruiting or modulating the RNA polymerase machinery and its associated factors.

Transcription is synthesis of RNA using one DNA strand as a template by an RNA polymerase. During elongation the RNA polymerase reads that template $3^{\prime }\to 5^{\prime }$ while extending RNA $5^{\prime }\to 3^{\prime }$. Only the selected region is transcribed; other regions need not be active, and the template strand can differ between genes. See Alberts et al.: RNA polymerase and selection of transcribed DNA regions.

For the displayed example, align these three nucleotide sequences in the same left-to-right positions:The first template triplet TAC selects AUG: T pairs with A, A with U, and C with G. The next CGA selects GCU, and ATT selects UAA. The coding strand therefore matches the RNA lettering except for T versus U, whereas the template is complementary. Reading that template in its own $5^{\prime }\to 3^{\prime }$ direction gives $5^{\prime }\text{-}\mathrm{TTAAGCCAT}\text{-}{3}^{\prime }$; orientation cannot be discarded. The gene expression diagram keeps all three rows aligned. See Clark, Douglas and Choi, Biology 2e: template and coding strands in transcription.

This correspondence makes nucleotide sequences interpretable, but replacing T by U is valid only after identifying the coding strand. Moreover, RNA may be processed after transcription, so the initial product and mature messenger RNA need not have identical uninterrupted nucleotide sequences. Transcription also makes functional RNA that will not undergo biological translation. See Alberts et al.: RNA processing and RNA as the final gene product.

The locally opened region of DNA in which RNA polymerase exposes a template for RNA synthesis. Its stability depends on base pairing, stacking and interactions with the enzyme; dye separation in a FRET reporter depends on the labelling geometry and need not change with the same sign for every reporter.

A regulatory factor that helps a transcription activator increase target gene expression, often through interactions with chromatin or the RNA polymerase machinery.

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.