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Pyrophosphate (PPi) off‐rate from an E. coli RNA polymerase (RNAP) elongation complex in the absence of nucleotides
Author(s) -
Johnson Ronald Sanders,
Strausbauch Mark,
Register J. Kristen
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.784.1
Subject(s) - nucleotide , pyrophosphate , rna polymerase , chemistry , biochemistry , elongation , transcription (linguistics) , rna , elongation factor , biophysics , polymerase , dna , biology , enzyme , ribosome , gene , materials science , ultimate tensile strength , metallurgy , linguistics , philosophy
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.