A NEW CLASS OF ACTIVATION-DEFECTIVE TATA-BINDING PROTEIN MUTANTS - EVIDENCE FOR 2 STEPS OF TRANSCRIPTIONAL ACTIVATION IN-VIVO

Using a genetic screen, we isolated four TATA-binding protein (TBP) mu tants that are specifically defective in vivo for the response to acid ic activators. In contrast to previously described activation-defectiv e TBP mutants, these TBP derivatives are not specifically defective fo r interactions with TATA elements or TFIIA, Three of these derivatives interact normally with a TATA element, TFIIA, TFIIB, or an acidic act ivation domain; presumably, they affect another protein-protein intera ction important for transcriptional activation, The remaining derivati ve (with F-237 replaced by D) binds a TATA element with wild-type affi nity, but the TBP-TATA complex has an altered electrophoretic mobility and interacts poorly with TFIIA and TFIIB; this suggests that the con formation of the TBP-TATA element complex plays a role in transcriptio nal activation, To determine the step at which the TBP derivatives wer e unable to activate transcription, we utilized an artificial recruitm ent assay in which TBP is targeted to the promoter via fusion to the L exA DNA-binding domain. Consistent with previous evidence that acidic activators can increase recruitment of TBP to the promoter in vivo, th e activation defect of some of these TBP derivatives can be corrected by artificial recruitment, In contrast, the activation defect of the o ther TBP derivatives is not bypassed by artificial recruitment. Thus, these TBP mutants define two steps in the process of transcriptional s timulation by acidic activators: efficient recruitment to the TATA ele ment and a postrecruitment interaction with a component(s) of the init iation complex.