Changes in the 17 bp spacer in the PR promoter of bacteriophage λ affect steps in open complex formation that precede DNA strand separation

Abstract

Tau plots and temperature-shift experiments were used to determine which step in the formation of transcriptionally-competent open complexes is affected by changing the length of the 17 bp spacer separating the −10 and −35 consensus regions of the PR promoter of bacteriophage λ. Abortive initiation assays at 37 °C indicate that the primary effect of insertion of a base-pair, thereby increasing spacer length to 18 bp, is a decrease in kf, the rate constant for conversion from closed (RPc) to open (RPo) complexes, by approximately a factor of 4. The mutation did not significantly affect KB, the equilibrium constant for formation of closed complexes, and decreased KBkf by a factor of 3. Deletion of a bp to create a 16 bp spacer had a much greater effect, decreasing the measured value of kf by a factor of about 25 to 30, and KBkf by a factor of 7 to 8. When the values of the parameters for the deletion mutant were corrected for incomplete occupancy of RPo at equilibrium, the effects of the deletion were even greater. In particular, the corrected value of KBkf was about 15 times lower than the corresponding value for two promoters with wild-type spacing.Based on temperature shift experiments, the changes in spacer length did not affect the equilibrium at 20 °C between RPi, a stable intermediate in which DNA strands are not separated, and RPo. Although differential sensitivity of single-stranded bases to KMnO4 indicated that in about 20 % of the open complexes at 20 °C the DNA strands are not fully separated (RPo1), the distribution between these complexes and RPo2 (DNA strands fully separated) was also not affected significantly by changes in spacer length. Thus, changes in spacer length primarily affect k2, the rate constant for conversion of RPc to RPi, which corresponds to a nucleation of DNA strand-separation.Application of published data and/or algorithms for determining effects of nucleotide sequence on twist angle or rise at individual bp steps does not provide a simple explanation of the difference in promoter strength between PR derivatives with 16 bp spacing and those with 18 bp spacing.