Figure 4.

Abortive initiation and promoter escape by RNAPs with mutations in region σ3.2. Transcription was performed on the T7A1cons-promoter template in the presence of the CpA primer, 25 µM ATP, GTP, CTP and 5 µM UTP; heparin was added to 10 µg/ml together with NTPs to prevent re-initiation. Positions of abortive and run-off (RO) RNA products are indicated; to resolve abortive and full-length RNAs, the samples were separated on gels of various concentrations (see ‘Materials and Methods’ section for details). The relative efficiencies of the run-off RNA synthesis are shown above the gel (averages and standard deviations from three independent experiments). Scanned profiles of abortive products (normalized by the amounts of 15 nt RNAs) are shown on the sides of the gel. Left, profiles for wild-type (WT), 513–516A and 522A σ⁷⁰ subunits; right, profiles for WT, Δ513–519 and Δ507–519 σ⁷⁰ subunits.