Oriented DNA binding by one-armed λ repressor heterodimers and contacts between repressor and RNA polymerase at P(RM)

Abstract

Bacteriophage λ repressor activates transcription from P(RM) by contacting the σ subunit of E. coli RNA polymerase. Although mutations in repressors that are defective in activation affect exposed residues in the repressor-operator co-crystal, the subunit in repressor dimers that is responsible for activation has not been determined experimentally. Here, we describe an oriented heterodimer approach using one-armed repressor-leucine zipper fusion proteins to resolve this question. Protection against methylation of operator DNA in vitro by one-armed and two-armed repressor- leucine zipper heterodimers shows that the subunit containing the arm in a one-armed repressor heterodimer binds preferentially in the consensus half- site of λ operators. The patterns of activation of the PRM promoter in vivo by one-armed heterodimers containing the pc2 mutation in only one subunit show that RNA polymerase at P(RM) contacts the N-terminal domain of the promoter-proximal subunit of the repressor dimer.