FIG. 2.

RNA Pol II-dependent transcription in egg extract. (A) Both low-speed supernatant (LSS) and high-speed supernatant (HSS) egg extract contain a functional transcription machinery (lanes 1 and 2) that is dependent on RNA Pol II as assessed by sensitivity to low levels (10 μg/ml) of α-amanitin (lanes 3 and 4). Except for lane 2, all in vitro transcription analysis in this study was performed with LSS extract. “H2B” indicates the position primer extension products corresponding to transcripts specifically initiated from a Xenopus histone H2B promoter, and the asterisk indicates the position of the primer extension product of synthetic LucΔ RNA, which was used as internal standard. Incubation of 0.6 μg of pH2B-Luc (160 fmol of DNA) with 1 μl of LSS egg extract under the conditions described in Materials and Methods results in the synthesis of ca. 0.2 to 0.5 fmol of correctly initiated transcript. (B) Two-dimensional gel electrophoresis in the presence of chloroquine. Panel B1 shows the DNA topology under standard egg extract in vitro transcription conditions, panel B2 shows a control sample containing nucleosomes, panel B3 explains the hybridization signals (“Rxd,” “Scl,” “Lnr,” and “NC” represent, respectively, relaxed, supercoiled, linearized, and nicked pH2B-Luc plasmid), panel B4 shows the positions of plasmid DNA treated with recombinant topoisomerase I (resulting in nicked and relaxed DNA), panel B5 shows the topology of plasmid as isolated from Escherichia coli, and panel B6 shows the position of linearized pH2B-Luc plasmid. The estimated average number of supercoils of the DNA in the in vitro transcription reaction, based on quantitated PhosphorImager area profiles of resolved DNA isomers, is 3.9. The uncertainty in the number of supercoils as a result of a dynamic equilibrium between supercoiling (writhe) and winding (twist) is estimated to be ±2 (see panel B4).