Figure 5.

Stalling affects the k_elong during transcription less than during replication. (A) Schematic representation of φ6 RdRP transcription with stalling. The transcription template was synthesized by hybridizing three ssRNAs. The single-stranded sequence transcribed prior stalling is positioned at the 3′-end of the template strand. φ6 RdRP is stalled with three NTPs at temperature T₁, and transcription reinitiated by the addition of UTP at temperature T₂. When the reaction is complete, Products A and B result. (B) Agarose gel analysis of aliquots collected at different time points after the reinitiation of the stalled φ6EC. The transcription template, Product A and Product B bands are indicated. Transcription intermediates have the lowest electrophoretic mobility and can be observed at 2′, 4′, 6′, 8′ and 10′ after reinitiation. (C) Transcription by φ6 RdRP in the absence of stalling. Aliquots collected at different polymerization times were analyzed on agarose gel. (D) An Arrhenius plot of the experimentally-observed elongation (red circles) and polymerization rates (black squares) of the reinitiated φ6EC and randomly initiated φ6 RdRP transcription, respectively, during the transcription reaction.