Purification and base composition analysis of phage lambda early promoters

Abstract

RNA-polymerase of Escherichia coli was allowed to bind to DNA of phage lambda in the absence of precursors. The resulting complex was excised by nuclease digestion and the protected DNA was recovered by phenol-extraction and ethanol precipitation. Acrylamide gel electrophoresis of protected DNA fragments reveals the existence of two distinct oligonucleotide peaks corresponding, respectively, to 45-52 and 7-10 nucleotide residues along with species of intermediate sizes. Peak I molecules have two properties: (a) their existence is dependent on the presence of sigma factor during the initial binding step, and (b) they are considerably enriched in A-T (up to 67%). On the contrary, peak II molecules have the same base composition as DNA of phage lambda, whether obtained in the presence or absence of sigma factor. Peak I molecules are thus believed to contain DNA sequences involved in promoter recognition, whether they are the promoters themselves, adjacent, or related sequences.