Changes in the 17 bp spacer in the P-R promoter of bacteriophage lambda affect steps in open complex formation that precede DNA strand separation

Tau plots and temperature-shift experiments were used to determine which st ep in the formation of transcriptionally-competent open complexes is affect ed by changing the length of the 17 bp spacer separating the -10 and -35 co nsensus regions of the P-R promoter of bacteriophage lambda. Abortive initi ation assays at 37 degrees C indicate that the primary effect of insertion of;a base-pair, thereby increasing spacer length to 18 bp, is a decrease in k(f), the rate constant for conversion from closed (RPc) to open (RPo) com plexes, by approximately a factor of 4. The mutation did not significantly affect K-B, the equilibrium constant for formation of closed complexes, and decreased K(B)k(f) by a factor of 3. Deletion of a bp to create a 16 bp sp acer had a much greater effect, decreasing the measured value of k(f) by a factor of about 25 to 30, and K(B)k(f) by a factor of 7 to 8. When the valu es of the parameters for the deletion mutant were corrected for incomplete occupancy of RP, at equilibrium, the effects of the deletion were even grea ter. In particular, the corrected value of K(B)k(f) was about 15 times lowe r than the corresponding value for two promoters with wild-type spacing. Based on temperature shift experiments, the changes in spacer length did no t affect the equilibrium at 20 degrees C between RPi, a stable intermediate in which DNA strands are not separated, and RPo. Although differential sen sitivity of single-stranded bases to KMnO4 indicated that in about 20% of t he open complexes at 20 degrees 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 k(2), the rate constant f or conversion of RPc to RPi, which corresponds to a nucleation of DNA stran d-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 P-R derivatives with 16 bp spacing and those with 18 bp spacing. (C) 2000 Academic Press.