Sequence Tolerance of the Phage λ P _RM Promoter: Implications for Evolution of Gene Regulatory Circuitry

Abstract

ABSTRACT Much of the gene regulatory circuitry of phage λ centers on a complex region called the O R region. This ∼100-bp region is densely packed with regulatory sites, including two promoters and three repressor-binding sites. The dense packing of this region is likely to impose severe constraints on its ability to change during evolution, raising the question of how the specific arrangement of sites and their exact sequences could evolve to their present form. Here we ask whether the sequence of a cis -acting site can be widely varied while retaining its function; if it can, evolution could proceed by a larger number of paths. To help address this question, we developed aλ cloning vector that allowed us to clone fragments spanning the O R region. By using this vector, we carried out intensive mutagenesis of the P RM promoter, which drives expression of CI repressor and is activated by CI itself. We made a pool of fragments in which 8 of the 12 positions in the− 35 and −10 regions were randomized and cloned this pool into the vector, making a pool of P RM variant phage. About 10% of the P RM variants were able to lysogenize, suggesting that the λ regulatory circuitry is compatible with a wide range of P RM sequences. Analysis of several of these phages indicated a range of behaviors in prophage induction. Several isolates had induction properties similar to those of the wild type, and their promoters resembled the wild type in their responses to CI. We term this property of different sequences allowing roughly equivalent function “sequence tolerance ” and discuss its role in the evolution of gene regulatory circuitry.