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. 2019 Oct 7;116(45):22573–22582. doi: 10.1073/pnas.1905449116

Fig. 3.

Fig. 3.

Separation of the postinitiation complexes stalled at +49. (A) PIC was assembled with 4-fold excess pol II and TFIIF relative to G-less 49 template DNA and then combined with ATP, 3′-O-methyl GTP, 4-fold excess Cet1-Ceg1, and 8-fold excess Abd1 (relative to the DNA). Reactions were incubated for 20 min at 30 °C, combined with 2 mM AMP-PNP, and sedimented on a 10 to 40% glycerol gradient; ∼130 µL per fraction were isolated and then analyzed by SDS/PAGE. (B and C) Protein identification of the PIC in fraction 11 (B) and free pol II-TFIIF in fraction 15 (C). (D) PIC was assembled in the same manner as in A. Transcription was initiated by addition of ATP, CTP, 3′-O-methyl GTP, UTP, [α-32P]UTP, Cet1-Ceg1, and Abd1. The postinitiation complexes were combined with 2 mM AMP-PNP, sedimented on a gradient, and analyzed as in A. (E) RNA analysis of fractions isolated in D by denaturing Urea-PAGE. The transcripts (∼25 nt) from the second round of initiation are indicated by a blue line. (F–H) Protein identification of the EC+PIC and EC+ITC in fraction 8 (F), EC in fraction 15 (G), and ITC in fraction 11 (H). Asterisk in D, E, and H indicates the presence of the ITC.