The conserved B3 domain of VIVIPAROUS1 has a cooperative DNA binding activity.
The biochemical activities that underlie the genetically defined activator and repressor functions of the VIVIPAROUS1 (VP1) protein have resisted in vitro analysis. Here, we show that a glutathione S-transferase (GST) fusion protein, including only the highly conserved B3 domain of VP1, has a highly cooperative, sequence-specific DNA binding activity. GST fusion proteins that include larger regions of the VP1 protein have very low activity, indicating that removal of the flanking protein sequences is necessary to elicit DNA binding in vitro. DNA competition and DNase I footprinting analyses show that B3 binds specifically to the Sph element involved in VP1 activation of the C1 gene, whereas binding to the G-box-type VP1-responsive element is of low affinity and is nonspecific. Footprint analysis of the C1 promoter revealed that sequences flanking the core TCCATGCAT motif of Sph also contribute to the recognition of the Sph element in its native context. The salient features of the in vitro GST-B3 DNA interaction are in good agreement with the protein and DNA sequence requirements defined by the functional analyses of VP1 and VP1-responsive elements in maize cells.
Suzuki
M
M
Horticultural Sciences Department, University of Florida, Gainesville 32605, USA.
Kao
C Y
CY
McCarty
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DR
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D003850:Deoxyribonuclease I; D005982:Glutathione Transferase / Q000096:biosynthesis; D008969:Molecular Sequence Data; D010940:Plant Proteins; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015534:Trans-Activators; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*; D003313:Zea mays / Q000378:metabolism*
D015342:DNA Probes; D004268:DNA-Binding Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D005982:Glutathione Transferase; D003850:Deoxyribonuclease I