Fig. 7. Mapping of Crt1. (A) Domains of Crt1. Amino acids 1–40 are dispensable for Crt1 function (Huang et al., 1998). The locations of the proline-rich domain, 77–155; DNA-binding domain, 246–318; a conserved ‘B’ box and a region displaying homology to numerous yeast and metazoan genes, 411–579 are indicated in the diagram. (B) Mapping of the TFIID interaction region. A 50 µg aliquot of GST derivatives bound to glutathione–agarose beads was incubated with yeast whole-cell extracts, washed and eluted as described in Materials and methods. TFIID binding was detected by western blotting for TAF_II145p and TBP. (C) Mapping of the Ssn6–Tup1 interaction region. A 20 µg aliquot of GST derivatives bound to glutathione–agarose beads was incubated with in vitro translated Ssn6 and Tup1, washed and eluted as described in Materials and methods. The binding of Ssn6 and Tup1 was detected by fluorography of SDS–polyacrylamide gels. (D) Identification of the repression domain of Crt1. Plasmids expressing Crt1–LexA DNA-binding domain fusion proteins were transformed into BY4705 cells containing the reporter plasmid JK101 (Brent and Ptashne, 1985) and grown in liquid SC-raffinose medium. β-galactosidase activities were measured in protein extracts prepared from at least three independent isolates. The results shown are from the same experiment; however, the average fold repression and SEMs from three independent experiments were: LexA–Crt1(1–811), 16.5 ± 1.8; LexA–Crt1(1–240) 37.5 ± 3.2; LexA–Crt1(1–350) 21.4 ± 1.6.