Figure 4.
Comparison of RNAP pause times at O1 with and without positive supercoiling. (A) A DNA tether was mechanically unwound forming plectonemes prior to the addition of NTPs. Subsequent transcription introduced positive supercoils that annihilated the mechanically induced, negative supercoils and lengthened the tether to a maximum. Further transcription and positive supercoiling eventually produced plectonemes that contracted the tether length. The dashed gray curve indicates the extension of the DNA tether during progressive conversion of negative to positive plectonemes due to transcription by RNAP. (B) Representative observations of extension versus time were recorded during transcription without (gray) and with (blue and red) LacI on a template pre-loaded with negative supercoiling. The blue arrow indicates the time at which all four NTPs or NTPs + LacI were introduced. The two vertical black dashed lines circumscribe a pause by RNAP at the LacI-O1 operator complex. (C) Representative observations of elongation were recorded in the absence of pre-loaded supercoiling without (left panel, gray and black) or with (right panel, blue and red) LacI. On the left is a schematic representation of the DNA template (D) Pause times by RNAP at the LacI obstacle varied as a function of torque on the DNA. Torque values were calculated using Equation (1), except that Ceff was estimated in 100 mM [Na+] instead of 50 mM [K+]. Circles represent measured pauses, while crosses represent averages.