When nickel-NTA beads were used, 10 m m imidazole was included in the binding and washing buffers to eliminate nonspecific binding. In vitro-translated general transcription factors were mixed with FREAC-2/nickel-NTA beads, and in vitro-translated FREAC-2 peptides were mixed with TBP-Sepharose beads in 50 μl of 20 m m Tris-Cl, pH 7.4, 100 m m NaCl, 5 m m MgCl 2, 10% glycerol, 0.1% Nonidet P-40, 2 m m β-mercaptoethanol, 0.1 mg/ml bovine serum albumin. Supernatants were diluted 5-fold with H 2O and assayed for FREAC-2 or Gal4 DNA binding activities in gel shifts (Ĭoupled in vitro transcription-translation of general transcription factors and parts of FREAC-2 was performed with TNT-T7 (Promega) and methionine (Amersham Pharmacia Biotech). The contents of the wells were transferred to 1.5-ml tubes and cleared by centrifugation. The plates were put back on ice, and 30 μl of nuclear extraction buffer (100 m mHepes, pH 7.9, 500 m m KCl, 25 m mMgCl 2, 35% glycerol, 10 m m dithiothreitol, protease inhibitors) was added to each well. The plates were tilted, and the cytoplasmic extract was carefully removed, without disturbing the nuclei attached to the bottom of the well, and assayed for luciferase activity. When extracts for gel shift assays should be made from the nuclei after the luciferase assay, protease inhibitors (20 μg/ml each of leupeptin, chymostatin and aprotinin 5 μg/ml each of pepstatin A and antipain 1 m mphenylmethylsulfonyl fluoride) were included in the cell lysis buffer. Cell lysis buffer (25 m m Tris-phosphate, pH 7.8, 2 m mdithiothreitol, 2 m m1,2-diaminocyclohexane- N,N,N′ ,N′-tetraacetic acid, 10% glycerol, 1% Triton X-100) was added (50 μl/well), and the plates were incubated on ice for 15 min.
Two days posttransfection, the medium was removed, and the cells were rinsed once with 0.5× Tris-buffered saline (1× Tris-buffered saline is 50 m m Tris-Cl, pH 7.8, 130 m m NaCl, 10 m m KCl, 5 m m MgCl 2). ) in 24-well tissue culture plates using Lipofectin or LipofectAMINE (Life Technologies, Inc.). Nuclear localization of FREAC-2 was found to depend on sequences from both ends of the forkhead domain. Overexpression of TFIIB potentiates activation by FREAC-2 in a manner dependent on the FREAC-2 activation domains. This sequence is well conserved among forkhead proteins, raising the possibility that interaction with TBP may be a general characteristic of this family of transcription factors. The part of FREAC-2 that binds TBP was mapped to 21 amino acids in the C-terminal end of the forkhead domain. TFIIB binds FREAC-2 close to the cleft between its two globular domains. The target site for FREAC-2 on TBP was localized to the N-terminal repeat in the core domain of TBP. FREAC-2 was shown to interact in vitro with TBP and TFIIB. Activation domain 2 is built up by three synergistic subdomains in the central part of the FREAC-2 protein. Activation domain 1 consists of the most C-terminal 23 amino acids of FREAC-2 and contains a sequence motif conserved in an activation domain of another forkhead protein, FREAC-1. We have identified the parts of FREAC-2 responsible for trans-activation and found two functionally redundant activation domains on the C-terminal side of the DNA binding forkhead domain.
#Freac covers Activator#
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