A TRANSCRIPTIONAL ACTIVATING REGION WITH 2 CONTRASTING MODES OF PROTEIN-INTERACTION

A C-terminal segment of the yeast activator Gal4 manifests two functio ns: When tethered to DNA, it elicits gene activation, and it binds the inhibitor Gal80. Here we examine the effects on these two functions o f cysteine and proline substitutions. We find that, although certain c ysteine substitutions diminish interaction with Gal80, those substitut ions have little effect on the activating function in vivo and interac tion with TATA box-binding protein (TBP) in vitro. Praline substitutio ns introduced near residues critical for Gal80 binding abolish that in teraction but once again have no effect on the activating function. Cr osslinking experiments show that a defined position in the activating peptide is in close proximity to TBP and Gal80 in the two separate rea ctions and show that binding of the inhibitor blacks binding to TBP. T hus, the same stretch of amino acids are involved in two quite differe nt protein-protein interactions: binding to Gal80, which depends on a precise sequence and the formation of a defined secondary structure, o r interactions with the transcriptional machinery in vivo, which are n ot impaired by perturbations of either sequence or structure.