Genetic analysis of the bacteriophage T4-encoded cochaperonin Gp31

Previous genetic and biochemical analyses have established that the bacteri ophage T4-encoded Gp31 is a cochaperonin that interacts with Escherichia co li's GroEL to ensure the timely and accurate folding of Gp23, the bacteriop hage-encoded major capsid protein. The heptameric Gp31 cochaperonin, like t he E. coli GroES cochaperonin, interacts with GroEL primarily through its u nstructured mobile loop segment. Upon binding to GroEL, the mobile loop ado pts a structured, beta-hairpin turn. In this article, we present extensive genetic data that strongly substantiate and extend these biochemical studie s. These studies begin with the isolation of mutations in gene 31 based on the ability to plaque on groEL44 mutant bacteria, whose mutant product inte racts weakly with Gp31. Our genetic system is unique because it also allows for the direct selection of revertants of such gene 31 mutations, based on their ability to plaque on groEL515 mutant bacteria. Interestingly, all of these revertants are pseudorevertants because the original 31 mutation is maintained. In addition, we show that the classical tsA70 mutation in gene 31 changes a conserved hydrophobic residue in the mobile loop to a hydrophi lic one. Pseudorevertants of tsA70, which enable growth at the restrictive temperatures, acquire the same mutation previously shown to allow plaque fo rmation on groEL44 mutant bacteria. Our genetic analyses highlight the cruc ial importance of all three highly conserved hydrophobic residues of the mo bile loop of Gp31 in the productive interaction with GroEL.