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 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 P(R) promoter of bacteriophage lambda. Abortive initiation 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 (RP(c)) to open (RP(o)) complexes, 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 spacer 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 values of the parameters for the deletion mutant were corrected for incomplete occupancy of RP(o) at equilibrium, the effects of the deletion were even greater. In particular, the corrected value of K(B)k(f) 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 degrees C between RP(i), a stable intermediate in which DNA strands are not separated, and RP(o). Although differential sensitivity of single-stranded bases to KMnO(4) indicated that in about 20% of the open complexes at 20 degrees C the DNA strands are not fully separated (RP(o1)), the distribution between these complexes and RP(o2) (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 for conversion of RP(c) to RP(i), 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 P(R) derivatives with 16 bp spacing and those with 18 bp spacing.
McKane
M
M
Department of Biological Sciences, University of Iowa, Iowa City, IA, 52246, USA.
Gussin
G N
GN
D000465:Algorithms; D010582:Bacteriophage lambda / Q000235:genetics*; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D018983:DNA Footprinting; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005814:Genes, Viral / Q000235:genetics*; D006493:Heparin / Q000494:pharmacology; D007536:Isomerism; D007700:Kinetics; D008957:Models, Genetic; D016254:Mutagenesis, Insertional / Q000235:genetics; D009691:Nucleic Acid Denaturation / Q000235:genetics; D011196:Potassium Permanganate / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D017384:Sequence Deletion / Q000235:genetics; D013696:Temperature; D013698:Templates, Genetic; D013816:Thermodynamics; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D012367:RNA, Viral; D011196:Potassium Permanganate; D006493:Heparin