title: Molecular structure of the GARP family of plant Myb-related DNA binding motifs of the Arabidopsis response regulators.
abstract: The B motif is a signature of type-B response regulators (ARRs) involved in His-to-Asp phosphorelay signal transduction systems in Arabidopsis. Homologous motifs occur widely in the GARP family of plant transcription factors. To gain general insight into the structure and function of B motifs (or GARP motifs), we characterized the B motif derived from a representative ARR, ARR10, which led to a number of intriguing findings. First, the B motif of ARR10 (named ARR10-B and extending from Thr-179 to Ser-242) possesses a nuclear localization signal, as indicated by the intracellular localization of a green fluorescent protein-ARR10-B fusion protein in onion epidermal cells. Second, the purified ARR10-B molecule binds specifically in vitro to DNA with the core sequence AGATT. This was demonstrated by several in vitro approaches, including PCR-assisted DNA binding site selection, gel retardation assays, and surface plasmon resonance analysis. Finally, the three-dimensional structure of ARR10-B in solution was determined by NMR spectroscopy, showing that it contains a helix-turn-helix structure. Furthermore, the mode of interaction between ARR10-B and the target DNA was assessed extensively by NMR spectroscopy. Together, these results lead us to propose that the mechanism of DNA recognition by ARR10-B is essentially the same as that of homeodomains. We conclude that the B motif is a multifunctional domain responsible for both nuclear localization and DNA binding and suggest that these insights could be applicable generally to the large GARP family of plant transcription factors.
authors.affiliation: Biochemistry Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Hosoda
authors.forename: Aya
authors.initials: A
authors.lastname: Imamura
authors.forename: Etsuko
authors.initials: E
authors.lastname: Katoh
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Hatta
authors.forename: Mari
authors.initials: M
authors.lastname: Tachiki
authors.forename: Hisami
authors.initials: H
authors.lastname: Yamada
authors.forename: Takeshi
authors.initials: T
authors.lastname: Mizuno
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Yamazaki
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D020598:Proto-Oncogene Proteins c-myb / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology
chemical_list: C469257:ARR10 protein, Arabidopsis; C511988:ATR1 protein, Arabidopsis; D029681:Arabidopsis Proteins; C511989:CPC protein, Arabidopsis; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D010940:Plant Proteins; D020598:Proto-Oncogene Proteins c-myb; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins