M Velleman

ban Operon of bacteriophage P1: Mutational analysis of the c1 repressor-controlled operator

The repressor of bacteriophage P1, encoded by the c1 gene, represses the phage lytic functions and is responsible for maintaining the P1 prophage in the lysogenic state. The c1 repressor interacts with at least 11 binding sites or operators widely scattered over the P1 genome. From these operators, a 17 base-pair asymmetric consensus sequence, ATTGCTCTAATAAATTT, was derived. Here, we show that the operator, Op72 of the P1ban operon consists of two overlapping 17 base-pair sequences a and b forming an incomplete palindrome. Op72a matches the consensus sequence, whereas Op72b contains two mismatches. The evidence is based on the sequence analysis of 27 operator mutants constitutive for ban expression. They were identified as single-base substitutions at positions 2 to 10 of Op72a (26 mutants) and at position 8 of Op72b (one mutant). We conclude from gel retardation and footprinting studies that two repressor molecules bind to the operator and that positions 4, 5 and 7 to 10 of the operator play an essential role in repressor recognition.

Purification of the C1 repressor of bacteriophage P1 by fast protein liquid chromatography

A fast protein liquid chromatographic method is described for the purification of the C1 repressor of bacteriophage P1 and its truncated form C1*. By using one crude extract, both repressor proteins were purified in parallel to homogeneity and were shown to interact specifically with P1 operator DNA in vitro. The method involves an affinity chromatographic step on heparin-Sepharose, followed by a combination of ion-exchange chromatography on Q Sepharose and S Sepharose. The availability of a homogeneous preparation of the phage repressor is a prerequisite for studies on its structure-function relationship.