Premium
Pyrophosphate (PPi) release during transcription elongation by E. coli RNA polymerase (RNAP)
Author(s) -
Johnson Ronald Sanders,
Strausbauch Mark
Publication year - 2007
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.21.5.a656-a
Subject(s) - processivity , nucleotide , elongation , chemistry , rna polymerase , dna , biophysics , polymerase , transcription (linguistics) , biochemistry , pyrophosphate , oligonucleotide , rna , microbiology and biotechnology , biology , enzyme , gene , materials science , linguistics , philosophy , ultimate tensile strength , metallurgy
Incorporation of nucleotides during transcription is accompanied by PP i production. Previous studies indicate that PP i in the active site blocks RNAP translocation. Thus, PP i release is a crucial step during elongation. In this study, synthetic elongation complexes (ECs) were used to directly measure PP i release with the Molecular Probes PP i assay kit (E‐6645) in stopped‐flow kinetic studies. The ECs (assembled and purified by established protocols) contained the core polymerase in a complex with a 9mer RNA primer annealed to a 30mer template DNA oligo that was associated with a fully complementary 30mer non‐template DNA oligo. The ECs were functional and displayed processivity. In these ECs, the next nucleotide to be incorporated in the sequence is UMP followed by AMP. Under conditions of ATP and UTP at 1 mM each in the presence of the ECs (100 nM), PP i release was complete in less than 50 milliseconds. In stark contrast, in the presence of only UTP (1 mM) along with the ECs (100 nM), there was no measurable PP i release in a 50 millisecond time‐base. It should be noted that after incorporation of UMP into the transcript, UTP becomes the incorrect nucleotide at the next step. These data are consistent with a model of elongation in which the binding of the correct nucleotide (presumably in the entry or preinsertion site) triggers rapid PP i release followed by RNAP translocation.