lac Repressor Fragments Produced In Vivo and In Vitro: An Approach to the Understanding of the Interaction of Repressor and DNA

Abstract

INTRODUCTION
Lac repressor is a tetrameric molecule with two types of binding sites. One site recognizes specifically the lac operator DNA; the other recognizes the inducer molecule, the small sugar derivative, isopropyl-β-D-thiogalactoside (IPTG). Although the amino acid sequence of the lac repressor and the DNA sequence of the operator are known (Beyreuther et al. 1973; Gilbert and Maxam 1973), our understanding of the direct molecular interactions of the two macromolecules is still rather poor. Among the different approaches to this problem is one which we specifically want to stress in this paper. It is based on the existence of lac repressor derivatives, obtained both in vivo and in vitro, which are missing the amino-terminal part of the molecule but still show the inducer-binding activity and the tetrameric oligomerization typical of lac repressor. These derivatives have been found in the past to be extremely useful in studying structure and function relationship in the lac repressor molecule.

We first encountered the in vivo derivatives during a collaboration with J. Miller on the protein-chemical characteristics of early nonsense mutations in the I gene. These mutations showed negative complementation and therefore were excellent candidates for studying the phenomenon of translational reinitiation in vivo. Indeed, we found that early nonsense mutations give rise to a reinitiation of translation past the amber block. The resulting mutationally altered proteins, which were missing the amino-terminal part of the polypeptide, could still form tetramers and display unimpaired inducer binding but were devoid of DNA binding (Platt et al. 1972;Ganem...