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

Abstract

Nucleotide incorporation during transcription is accompanied by PPi formation. Because PPi at the active site blocks RNAP translocation, its release is a crucial step in transcription. Previously, we found that rapid PPi release after nucleotide incorporation only occurred if the next nucleotide required for incorporation was present. As an extension of these studies, we investigated PPi 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 PPi 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 PPi concentration was hyperbolic. Data analysis indicated that the rate constant for PPi 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 PPi followed by RNAP translocation.