Chemical synthesis and biochemical reactivity of bacteriophage lambda PR promoter.
By a combination of chemical and enzymatic methods, a 75 base pair DNA duplex containing the sequence of the lambda PR promoter including the OR1 and OR2 cI repressor binding sites was synthesized. The solid support phosphite triester procedure (Caruthers, M. H. et al., Cold Spring Harbor Symposia on Quantitative Biology XLVII, in press) was used for the synthesis of oligonucleotides comprising the sequence. We report here an adaptation of the method of DNA synthesis in test tubes. Assembly of the oligonucleotides involved the use of T4 polynucleotide kinase and T4 DNA ligase. We show that the synthetic DNA is recognized by RNA polymerase and cI repressor in a manner identical to the same control region contained on a restriction fragment isolated from bacteriophage lambda DNA. Our synthetic approach using chemically synthesized promoter variants is thus suitable for studies probing the function of promoters.
D010582:Bacteriophage lambda / Q000235:genetics*; D001482:Base Composition; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000138:chemical synthesis* / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D009876:Operon
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes