Open AccessInteraction of RNA Polymerase with Promoters from Bacteriophage fd
Author(s) -
SEEBURG Peter H.,
NÜSSLEIN Christiane,
SCHALLER Heinz
Publication year - 1977
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1977.tb11372.x
Subject(s) - promoter , transcription preinitiation complex , rna polymerase , dna , base pair , restriction enzyme , transcription (linguistics) , abortive initiation , microbiology and biotechnology , transcription factor ii e , bacteriophage , rna , chemistry , polymerase , biology , escherichia coli , biophysics , rna dependent rna polymerase , biochemistry , gene , gene expression , linguistics , philosophy
Replicative form DNA of bacteriophage fd, which had been fragmented with the restriction endonuclease II from Hemophilus parainfluenzae (endo R · Hpa II), was reacted with Escherichia coli RNA polymerase; the resulting stable preinitiation complexes were analysed using the filter binding assay followed by gel electrophoresis. At 120 mM KCl the first‐order rate constants for complex decay were determined to be 10 −2 –10 −6 s −1 . The second‐order rate constants for complex formation were found to be about 10 6 –10 7 M −1 s −1 . From these values association constants for the individual promoters were calculated to be 2 × 10 −8 –2 × 10 −11 M −1 . The rate of formation and the stability of promoter complexes was enhanced in superhelical DNA. No evidence was found for stable promoter‐specific closed complexes consisting of enzyme and helical DNA. This and the kinetic data suggest that the unwinding of base pairs is already important early in promoter selection, and not only for the formation of the final open complex. The initiation of RNA synthesis from the preinitiation complex was faster than complex dissociation and complex formation for all promoters. Consequently, the initiation efficiency of a promoter is determined by the rate of complex formation, and not by its ‘affinity’ for the enzyme. No correlation was found between the relative order of the fd promoters for the binding and the dissociation reaction. This is explained by different structural determinants, for the two reactions, which are located in different parts of the promoter DNA.