ABSTRACT
The TATA binding protein (TBP) is a central component of the eukaryotic transcription machinery and is subjected to both positive and negative regulation. As is evident from structural and functional studies, TBP's concave DNA binding surface is inhibited by a number of potential mechanisms, including homodimerization and binding to the TAND domain of the TFIID subunit TAF1 (yTAF(II)145/130). Here we further characterized these interactions by creating mutations at 24 amino acids within the Saccharomyces cerevisiae TBP crystallographic dimer interface. These mutants are impaired for dimerization, TAF1 TAND binding, and TATA binding to an extent that is consistent with the crystal or nuclear magnetic resonance structure of these or related interactions. In vivo, these mutants displayed a variety of phenotypes, the severity of which correlated with relative dimer instability in vitro. The phenotypes included a low steady-state level of the mutant TBP, transcriptional derepression, dominant slow growth (partial toxicity), and synthetic toxicity in combination with a deletion of the TAF1 TAND domain. These phenotypes cannot be accounted for by defective interactions with other known TBP inhibitors and likely reflect defects in TBP dimerization.
Subject(s)
Mutation , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , TATA-Box Binding Protein/chemistry , TATA-Box Binding Protein/genetics , Adenosine Triphosphatases , Binding Sites , Cell Division , Crystallography, X-Ray , DNA Helicases/genetics , Dimerization , Gene Expression Regulation, Fungal , Models, Molecular , Mutagenesis, Site-Directed , Phenotype , Protein Conformation , Protein Structure, Tertiary , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/metabolism , Structure-Activity Relationship , TATA Box , TATA-Binding Protein Associated Factors/genetics , TATA-Binding Protein Associated Factors/metabolism , TATA-Box Binding Protein/metabolism , Transcription Factor TFIID/metabolism , Transcription, GeneticABSTRACT
We have built a microarray database, StressDB, for management of microarray data from our studies on stress-modulated genes in Arabidopsis. StressDB provides small user groups with a locally installable web-based relational microarray database. It has a simple and intuitive architecture and has been designed for cDNA microarray technology users. StressDB uses Windows 2000 as the centralized database server with Oracle 8i as the relational database management system. It allows users to manage microarray data and data-related biological information over the Internet using a web browser. The source-code is currently available on request from the authors and will soon be made freely available for downloading from our website athttp://arastressdb.cac.psu.edu.
