A new class of activation-defective TATA-binding protein mutants: evidence for two steps of transcriptional activation in vivo.
Using a genetic screen, we isolated four TATA-binding protein (TBP) mutants that are specifically defective in vivo for the response to acidic activators. In contrast to previously described activation-defective TBP mutants, these TBP derivatives are not specifically defective for interactions with TATA elements or TFIIA. Three of these derivatives interact normally with a TATA element, TFIIA, TFIIB, or an acidic activation domain; presumably, they affect another protein-protein interaction important for transcriptional activation. The remaining derivative (with F-237 replaced by D) binds a TATA element with wild-type affinity, but the TBP-TATA complex has an altered electrophoretic mobility and interacts poorly with TFIIA and TFIIB; this suggests that the conformation of the TBP-TATA element complex plays a role in transcriptional activation. To determine the step at which the TBP derivatives were unable to activate transcription, we utilized an artificial recruitment assay in which TBP is targeted to the promoter via fusion to the LexA DNA-binding domain. Consistent with previous evidence that acidic activators can increase recruitment of TBP to the promoter in vivo, the activation defect of some of these TBP derivatives can be corrected by artificial recruitment. In contrast, the activation defect of the other TBP derivatives is not bypassed by artificial recruitment. Thus, these TBP mutants define two steps in the process of transcriptional stimulation by acidic activators: efficient recruitment to the TATA element and a postrecruitment interaction with a component(s) of the initiation complex.
Stargell
L A
LA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Struhl
K
K
D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D005816:Genetic Complementation Test; D046911:Macromolecular Substances; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012320:RNA Polymerase III / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D035181:TATA-Box Binding Protein; D035561:Transcription Factor TFIIA; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D035181:TATA-Box Binding Protein; D035561:Transcription Factor TFIIA; D014157:Transcription Factors; D012320:RNA Polymerase III