title: Rapid pyrophosphate release from transcriptional elongation complexes appears to be coupled to a nucleotide-induced conformational change in E. coli core polymerase.
abstract: In the nucleotide addition cycle, pyrophosphate is generated upon incorporation of each nucleotide. Rapid release of pyrophosphate is essential for facile transcription elongation. Stopped-flow kinetic studies involving alterations in the intrinsic protein fluorescence of the core polymerase upon the binding of pyrophosphate to well-defined elongation complexes (ECs) indicate that the intrinsic off-rate of pyrophosphate (k=5.7-8.1 s(-1)) is too slow to account for the rapid rate of nucleotide incorporation that occurs during processive transcription elongation. Stopped-flow kinetic studies on UTP binding followed by UMP incorporation into an EC as monitored by alterations in the intrinsic protein fluorescence of the core polymerase resulted in a set of first-order rate constants that varied in a hyperbolic manner as a function of UTP concentration. This is consistent with a binding step (K(UTP)=17±6 μM) followed by a conformational change (k=623±54 s(-1)) in the core polymerase. In comparable studies on ATP binding and AMP incorporation into an EC, the data were also consistent with a binding step (K(ATP)=44±6 μM) followed by a conformational change (k=411±51 s(-1)) in the core polymerase. In stopped-flow kinetic studies with α,β-methyleneadenosine 5' triphosphate, which can bind to the EC but cannot lead to nucleotide incorporation, the analysis of the hyperbolic dependence of the observed first-order rate constant on α,β-methyleneadenosine 5' triphosphate concentration yielded a value of 20±13 μM for the apparent dissociation constant and a value of 221±36 s(-1) for the first-order rate constant for the associated conformational change in the core polymerase. This indicates that the conformational change in the core polymerase precedes chemistry. In conjunction with previously reported results on the increase in the rate of pyrophosphate release in the presence of the next cognate nucleotide for incorporation, the data are consistent with a model in which rapid pyrophosphate release is coupled to a conformational change in the core polymerase that precedes chemistry and that occurs upon the binding of the next cognate nucleotide for incorporation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC 27834, USA. johnsonro@ecu.edu
authors.forename: Ronald S
authors.initials: RS
authors.lastname: Johnson
authors.forename: Mark
authors.initials: M
authors.lastname: Strausbauch
authors.forename: J Kristen
authors.initials: JK
authors.lastname: Carraway
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D014158:Transcription, Genetic
chemical_list: D011756:Diphosphates; D009711:Nucleotides; D012321:DNA-Directed RNA Polymerases