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Pyrophosphate (PPi) suppression of transcription elongation as catalyzed by E. coli RNA polymerase is not mediated directly by product inhibition or pyrophosphorolysis
Author(s) -
Johnson Ronald S.,
Strausbauch Mark,
Carraway J. Kristen
Publication year - 2011
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.25.1_supplement.501.1
Subject(s) - elongation , pyrophosphate , transcription (linguistics) , nucleotide , rna polymerase , footprinting , chemistry , biochemistry , rna polymerase ii , microbiology and biotechnology , rna , elongation factor , biophysics , dna , biology , enzyme , gene expression , promoter , gene , base sequence , materials science , linguistics , philosophy , metallurgy , ultimate tensile strength , ribosome
We tested the hypothesis that suppression of transcription elongation by PPi is due to either product inhibition or pyrophosphorolysis by using well‐defined elongation complexes (EC). The effect of PPi on the elongation of the RNA from 9 to 10 nucleotides in EC30:RNA9 by the addition of UMP was monitored by gel electrophoresis. It was observed that there was little if any decrease in UMP incorporation at PPi concentrations of 1 and 5 mM PPi respectively in a 1 minute assay. Moreover, nearly identical rate constants were observed for UMP incorporation in the absence and presence of 1 mM PPi in quench flow experiments. Because pyrophosphorolysis is dependent on the core polymerase being in the pre‐translocated state, footprinting studies were conducted. Analysis of the data in the absence and presence of 1 mM PPi indicated that the predominant state is the pre‐translocated state in both cases. Thus, it appears that PPi suppression of transcription elongation is not mediated directly by product inhibition or pyrophosphorolysis. When transcription elongation was monitored in the presence of all four nucleotides in the absence and presence of 1 mM PP, it was observed that PPi increased the extent of pausing. This appears to be the mechanism by which PPi suppresses transcription elongation in this system. NSF Grant #211369