USE OF A 2-HYBRID SYSTEM TO IDENTIFY MUTATIONS IN MAX THAT CONFER INCREASED AFFINITY FOR MYC

A yeast two-hybrid system was used to identify mutants of Max that exh ibit an increased affinity for Myc, Truncated forms of the Max helix-l oop-helix/leucine zipper motif (HLH/Zip) were first expressed in a two -hybrid system in which the bait protein was the HLH/Zip motif of Myc, Deletion of amino acids both aminoterminal and carboxy-terminal to th e leucine zipper of Max reduced Myc/Max heterodimer formation as evide nced by a 160-fold reduction in the expression of the lacZ gene, A lib rary of partially randomized sequences encoding this minimal leucine z ipper of Max was then screened using the two-hybrid system, Mutant for ms of the Max leucine zipper were identified whose affinities for Myc, as measured by P-galactosidase activity in yeast lysates were from 8- to 200-fold greater than the wild-type Max zipper, These Max mutants were shown to interact specifically with Myc and not with wild-type Ma x. Of 29 mutants analyzed, all had a unique amino acid sequence. This result illustrates the value of a genetic screen in the identification of a collection of mutant forms of the Max leucine zipper whose struc tures would not have been predicted based on principles of structure-b ased design.