HOW DO ZN2CYS6 PROTEINS DISTINGUISH BETWEEN SIMILAR UPSTREAM ACTIVATION SITES - COMPARISON OF THE DNA-BINDING SPECIFICITY OF THE GAL4 PROTEIN IN-VITRO AND IN-VIVO

The GAL4 protein of Saccharomyces cerevisiae is the prototype of a fam ily of transcription factors that contain a ''Zn2Cys6'' coordination c omplex in the DNA-binding domain. GAL4 activates the transcription of genes involved in galactose and melibiose metabolism by binding to sit es that contain one or more copies of a sequence 5'-CGGN5TN5CCG-3'. Ot her Zn2Cys6 proteins in S. cerevisiae also recognize sequences contain ing two CGG triplets, but with different spacings between them. In thi s report we investigate the mechanism by which GAL4 distinguishes its bona fide binding site from similar sequences as well as from bulk gen omic DNA. In vitro, GAL4 recognizes with moderate to high affinity a v ariety of sites of the general formula (A/C)GGN10-12CCG. This level of specificity is apparently insufficient for the activator to carry out its biological role. However, many of the sites to which GAL4 binds i n vitro do not support GAL4-activated transcription in vivo. In most c ases there is not a quantitative correlation between the relative affi nity of a site for GAL4 in vitro and the level of GAL4-dependent trans cription supported by it in vivo. These data imply that there is some mechanism in vivo by which the intrinsic binding specificity of GAL4 i s modified.