DIRECT ACTIVATION AND ANTI-REPRESSION FUNCTIONS OF GAL4-VP16 USE DISTINCT MOLECULAR MECHANISMS

In order to determine whether the molecular mechanisms used for direct activation by GAL4-VP16 are the same as those used for anti-repressio n, we have employed monoclonal antibodies specific for the VP16 activa tion domain. In the absence of added repressors, GAL4-VP16 was able to stimulate transcription from a template containing GAL4-binding sites , and the antibodies raised against the VP16 activation domain failed to inhibit this direct activation. GAL4-VP16 also was able to prevent histone HI-mediated repression by a mechanism that was strongly depend ent on the presence of specific GAL4-binding elements in the promoter. However, in contrast to the assays conducted in the absence of repres sors, the antibodies were strong inhibitors of GAL4-VP 16-activated tr anscription in the presence of histone H1. Thus the binding of the ant ibodies distinguished between the direct activation and anti-repressio n functions of GAL4-VP16, indicating that these functions operate thro ugh distinct molecular mechanisms. The anti-repressionspecific mechani sm that is inhibitable by the antibodies acted at an early stage of pr einitiation complex formation. Deletions of individual subdomains of t he VP16 activation domain demonstrated that there was not a discrete s ubdomain responsible for the anti-repression function of GAL4-VP16. Th us, the inhibitory effect of the antibodies appeared to be due to the location of the epitope within the activator protein rather than to so me inherent biochemical property of that region of the protein that is required specifically for anti-repression. The inhibitory effect of t he antibodies also ruled out the possibility that steric exclusion of repressor proteins from the promoter was the sole means of anti-repres sion by the transcriptional activator.