Figure 6.

Charge heterogeneity of active and inactive forms of PTRF. (A) Diagram showing the chromatographic steps used to purify PTRF from nuclear extracts. (B) Chromatographic separation of release-competent and -incompetent forms of PTRF. Individual fractions from the last purification step, e.g., Mono Q_II, were analyzed on western blots or assayed for their capability to dissociate ternary transcription complexes. Ternary complexes were formed by pre-incubating bead-bound tailed template (pCAT-T6-T₁) for 5 min with Pol I, TTF-I and nucleotides to allow Pol I to reach the terminator. Paused complexes were removed by magnetic attraction, washed with buffer AM-200, and then incubated for another 5 min with cold nucleotides in the presence of fractions MQ_II #18 and #24, respectively. Transcripts were separated into template-bound (b) and released (r) fractions. 2D gel electrophoresis of fractions MQ_II #18 and #24. The two fractions were subjected to 2D gel electrophoresis, blotted onto nitrocellulose filters, and PTRF was immunostained with anti-PTRF antibodies.