Pyrophosphate (PPi) off‐rate from an E. coli RNA polymerase (RNAP) elongation complex in the absence of nucleotides

Nucleotide incorporation during transcription is accompanied by PP i formation. Because PP i at the active site blocks RNAP translocation, its release is a crucial step in transcription. Previously, we found that rapid PP i release after nucleotide incorporation only occurred if the next nucleotide required for incorporation was present. As an extension of these studies, we investigated PP i release from an elongation complex (EC) in the absence of nucleotides. The RNA primer in the EC contained a phosphorothioate internucleotide linkage at the 3′ end to block pyrophosphorolysis. The interaction of PP i with this EC was monitored by alterations in the intrinsic protein fluorescence in stopped‐flow kinetic studies. A plot of the observed first‐order rate constant against PP i concentration was hyperbolic. Data analysis indicated that the rate constant for PP i release (6.1±1.9 s −1 ) in the absence of NTPs was ∼100 fold less than that observed in the presence of the correct NTP for incorporation in previous studies. This supports a model for elongation in which the binding of the correct nucleotide (in either the entry or preinsertion site) triggers the rapid release of PP i followed by RNAP translocation.