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Tryptophan fluorescence quenching kinetic studies of steps of open complex formation by E. coli RNA polymerase (945.1)
Author(s) -
Xia Xue,
Murray Jan,
Record Thomas,
Yang Zao
Publication year - 2014
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.28.1_supplement.945.1
Subject(s) - kinetics , chemistry , quenching (fluorescence) , rna polymerase , biophysics , tryptophan , polymerase , dna , binding site , protein subunit , fluorescence , base pair , stereochemistry , rna , biochemistry , biology , physics , amino acid , quantum mechanics , gene
To initiate transcription, E. coli RNA polymerase (RNAP) binds promoter DNA, bends the start site region into the active site cleft, opens it using binding free energy, and places the start site base of the template strand in the active site. Structural studies of the open complex show that bending is accompanied by insertion of the ‐11 base into a pocket on the specificity (σ70) subunit. We are using stopped flow fluorescence measurements of tryptophan (W) quenching to investigate when in the mechanism of open complex formation this base‐flipping step occurs, and to characterize the kinetics of this step for different promoters for comparison with other kinetic assays for formation of closed and open complexes. For the λPR promoter at 42°C, tryptophan quenching kinetics fit two exponentials, as expected for the rapid‐equilibrium formation of an ensemble of closed complexes, including the base flipped species, followed by the slower irreversible opening step; rates of these steps are comparable to those determined by other assays. The kinetics are complicated by slower decays which appear to be photobleaching. Grant Funding Source : NIH GM103061