Two distinct domains in the yeast transcription factor IID and evidence for a TATA box-induced conformational change.
Transcription factor IID from Saccharomyces cerevisiae (YIID) binds the TATA box element present in most RNA polymerase II promoters. In this work, partial proteolysis was used as a biochemical probe of YIID structure. YIID consists of a protease-sensitive amino terminus and a highly stable, protease-resistant carboxy-terminal core. The cleavage sites of the predominant chymotrypsin- and trypsin-derived fragments were mapped to amino acid residues 40 to 41 and 48 to 49, respectively, by amino-terminal peptide sequencing. Removal of the amino terminus resulted in a dramatic increase in the ability of YIID to form a stable complex with DNA during gel electrophoresis mobility shift assays and a two- to fourfold increase in DNA-binding affinity, as assayed by DNase I footprinting analysis. The carboxy-terminal 190-amino-acid core was competent for transcription in vitro and was similar in activity to native YIID. DNA containing a TATA element induced hypersensitive sites in the amino-terminal domain and stabilized the core domain to further proteolytic attack. Native YIID did not bind to a TATA box at 0 degrees C, whereas the carboxy-terminal DNA-binding domain did. These results suggest that YIID undergoes a conformational change upon binding to a TATA box. Southern blotting showed that the carboxy-terminal domain is highly conserved, while the amino-terminal domain diverged rapidly in evolution, even between closely related budding yeasts.
Lieberman
P M
PM
Department of Microbiology, University of California, Los Angeles 90024-1570.
Schmidt
M C
MC
Kao
C C
CC
Berk
A J
AJ
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000648:ultrastructure*; D005656:Fungal Proteins / Q000648:ultrastructure; D015870:Gene Expression; D005800:Genes, Fungal; D066298:In Vitro Techniques; D015394:Molecular Structure; D010449:Peptide Mapping; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship; D013696:Temperature; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000648:ultrastructure*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D035362:Transcription Factor TFIID; D014157:Transcription Factors