Open AccessIn vitro comparison of initiation properties of bacteriophage lambda wild-type PR and x3 mutant promoters.
Author(s) -
Diane K. Hawley,
William R. McClure
Publication year - 1980
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.77.11.6381
Subject(s) - bacteriophage , promoter , mutant , rna polymerase , polymerase , microbiology and biotechnology , abortive initiation , biology , siphoviridae , lambda phage , in vitro , wild type , chemistry , rna , dna , genetics , escherichia coli , gene , gene expression
The in vitro initiation properties of the PR promoter of bacteriophage lambda and of a PR mutant, x3, were compared. Using the abortive initiation reaction, we measured the lags in the approach to a final steady-state rate when dinucleotide synthesis was initiated with RNA polymerase. These lags corresponded to the average times required for the formation of transcriptionally active open complexes. By measuring the lags at different RNA polymerase concentrations, we could separate open complex formation into two steps, based on a simple model in which the initial bimolecular association of free promoter and polymerase in a closed complex is followed by an isomerization to the open complex. The contribution of each step to the overall rate of open complex formation was quantitated for both promoters. We found that the x3 mutation, which is located in the -35 region of PR, resulted in a decrease in the association constant for the initial binding to the closed complex to 5% of its wild-type value and a decrease in the rate of the isomerization to 20%. The lifetimes and abortive initiation characteristics of the mutant and wild-type promoters were similar. We concluded that the main effect of the x3 mutation was to increase the average time of open complex formation and that the functional properties of the open complexes did not differ significantly between the two promoters.