title: Identification of the core domain and the secondary structure of the transcriptional coactivator MBF1.
abstract: BACKGROUND
  Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA binding activators, such as FTZ-F1 and GCN4. MBF1 bridges the DNA-binding regions of these activators and the TATA-box binding protein (TBP), suggesting that MBF1 functions by recruiting TBP to promoters where the activators are bound. In addition, MBF1 stimulates DNA binding activities of the activators to their recognition sites. To date, little is known about structures of coactivators that bind to TBP.
  
  
  RESULTS
  The two-dimensional (2D) 1H-15N correlation spectrum of 15N labeled MBF1 indicated that MBF1 consists of both flexible and well structured parts. Limited digestion of MBF1 by alpha-chymotrypsin yielded a approximately 9 kDa fragment. N-terminal sequence analysis and NMR measurements revealed that this fragment originates from the C-terminal 80 residues of MBF1 and form a well structured C-terminal domain of MBF1, MBF1CTD. As previous deletion analyses have shown that MBF1CTD is capable of binding to TBP, it is suggested that MBF1CTD is the TBP binding domain of MBF1. Sequential assignments have been obtained by means of three-dimensional (3D) and four dimensional (4D) heteronuclear correlation spectroscopies, and then the secondary structure of MBF1CTD was determined. As a result, MBF1CTD was shown to contain four amphipathic helices and a conserved C-terminal region. Asp106 which is assumed to be responsible for the binding to TBP is located at the hydrophilic side of the third helix.
  
  
  CONCLUSIONS
  Structural analyses revealed that MBF1 consists of two structurally different domains. A N-terminal region is indispensable for the binding to activators, and does not form a well defined structure. In contrast, the C-terminal 80 residues, which is capable of binding to TBP by itself, form a well-structured domain, MBF1CTD. MBF1CTD is made up of four amphipathic helices and a conserved C-terminal tail. A putative TBP binding residue is located on the hydrophilic surface of the third helix.
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Ozaki
authors.forename: K I
authors.initials: KI
authors.lastname: Takemaru
authors.forename: T
authors.initials: T
authors.lastname: Ikegami
authors.forename: M
authors.initials: M
authors.lastname: Mishima
authors.forename: H
authors.initials: H
authors.lastname: Ueda
authors.forename: S
authors.initials: S
authors.lastname: Hirose
authors.forename: Y
authors.initials: Y
authors.lastname: Kabe
authors.forename: H
authors.initials: H
authors.lastname: Handa
authors.forename: M
authors.initials: M
authors.lastname: Shirakawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D012831:Bombyx / Q000737:chemistry* / Q000235:genetics; D002918:Chymotrypsin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017433:Protein Structure, Secondary; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D013329:Structure-Activity Relationship; D035181:TATA-Box Binding Protein; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011522:Protons; D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D014157:Transcription Factors; D002918:Chymotrypsin