RESUMEN
Mot1 is an essential, conserved TATA-binding protein (TBP)-associated factor in Saccharomyces cerevisiae and a member of the Snf2/Swi2 ATPase family. Mot1 uses ATP hydrolysis to displace TBP from DNA, an activity that can be readily reconciled with its global role in gene repression. Less well understood is how Mot1 directly activates gene expression. It has been suggested that Mot1-mediated activation can occur by displacement of inactive TBP-containing complexes from promoters, thereby permitting assembly of functional transcription complexes. Mot1 may also activate transcription by other mechanisms that have not yet been defined. A gap in our understanding has been the absence of biochemical information related to the activity of Mot1 on natural target genes. Using URA1 as a model Mot1-activated promoter, we show striking differences in the way that both TBP and Mot1 interact with DNA compared with other model DNA substrates analyzed previously. These differences are due at least in part to the propensity of TBP alone to bind to the URA1 promoter in the wrong orientation to direct appropriate assembly of the URA1 preinitiation complex. The results suggest that Mot1-mediated activation of URA1 transcription involves at least two steps, one of which is the removal of TBP bound to the promoter in the opposite orientation required for URA1 transcription.
Asunto(s)
ADN Helicasas/metabolismo , ADN de Hongos/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Regiones Promotoras Genéticas/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Transcripción Genética/fisiología , Adenosina Trifosfatasas , Adenosina Trifosfato/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , ADN Helicasas/química , ADN Helicasas/genética , ADN de Hongos/química , ADN de Hongos/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Hidrólisis , Unión Proteica/fisiología , Estructura Cuaternaria de Proteína/fisiología , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Factores Asociados con la Proteína de Unión a TATA/química , Factores Asociados con la Proteína de Unión a TATA/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Recombinant full-length Saccharomyces cerevisiae TATA binding protein (TBP) and its isolated C-terminal conserved core domain (TBPc) were prepared with measured high specific DNA-binding activities. Direct, quantitative comparison of TATA box binding by TBP and TBPc reveals greater affinity by TBPc for either of two high-affinity sequences at several different experimental conditions. TBPc associates more rapidly than TBP to TATA box bearing DNA and dissociates more slowly. The structural origins of the thermodynamic and kinetic effects of the N-terminal domain on DNA binding by TBP were explored in comparative studies of TBPc and TBP by "protein footprinting" with hydroxyl radical (*OH) side chain oxidation. Some residues within TBPc and the C-terminal domain of TBP are comparably protected by DNA, consistent with solvent accessibility changes calculated from core domain crystal structures. In contrast, the reactivity of some residues located on the top surface and the DNA-binding saddle of the C-terminal domain differs between TBP and TBPc in both the presence and absence of bound DNA; these results are not predicted from the crystal structures. A strikingly different pattern of side chain oxidation is observed for TBP when a nonionic detergent is present. Taken together, these results are consistent with the N-terminal domain actively modulating TATA box binding by TBP and nonionic detergent modulating the interdomain interaction.
