Figure 2.

Minicircle transcription assays. (A) Looped DNA topologies containing a highly bent promoter (arrow) are recreated with DNA minicircles, eliminating any influence on transcription owing to either the presence of a repressor protein or variations in bending and torsional stresses within supercoiled DNA. The minicircle template contains a T7 RNAP promoter sequence, a site at which T7 RNAP ECs will stall in the absence of UTP (shaded octagon), and a target sequence (shaded arc) from which the corresponding RNA transcript will hybridize to a designed molecular beacon sequence. (B) T7 RNAP (E_free) is incubated with the DNA template without UTP, and forms stalled ECs (E_stalled) at some rate of complex formation (k_for). (C) Heparin addition inactivates free T7 RNAP (E_{free(inhibited)}). (D) UTP addition initiates transcription from active ECs (E_elong), which synthesize RNA at the elongation velocity (k_cat). (E) Molecular beacons hybridize to target transcript sequence. (F) Elongating T7 RNAP detach from the template at some rate of dissociation (k_diss). (G) A simplified kinetic equation governing T7 RNAP behavior in the assay.