Solution Structure of the B3 DNA Binding Domain of the Arabidopsis Cold-Responsive Transcription Factor RAV1[W]

Abstract

The B3 DNA binding domain is shared amongst various plant-specific transcription factors, including factors involved in auxin-regulated and abscisic acid-regulated transcription. Herein, we report the NMR solution structure of the B3 domain of the Arabidopsis thaliana cold-responsive transcription factor RAV1. The structure consists of a seven-stranded open b-barrel and two a-helices located at the ends of the barrel and is significantly similar to the structure of the noncatalytic DNA binding domain of the restriction enzyme EcoRII. An NMR titration experiment revealed a DNA recognition interface that enabled us to propose a structural model of the protein-DNA complex. The locations of the DNA-contacting residues are also likely to be similar to those of the EcoRII DNA binding domain.

Genes directly regulated by LEAFY COTYLEDON2 provide insight into the control of embryo maturation and somatic embryogenesis

“…Our demonstration that LEC2 binds with the same DNA element bound by FUS3 and ABI3 (30,(38)(39)(40), the RY motif, provides a partial explanation for similarities in the gain-of-function phenotypes. Consistent with this observation, LEC2, ABI3, and FUS3 share identical or conserved amino acid residues at positions in the B3 domain implicated as being responsible for DNA-binding specificity based on the solution structure of the B3 domain protein RAV1 (41). Thus, all three transcription factors bind RY motifs through their B3 domains and activate maturation-specific genes, although we note that ABI3 activity is influenced by an additional domain that binds ABA response elements (40).…”

The Arabidopsis B3 Domain Protein VERNALIZATION1 (VRN1) Is Involved in Processes Essential for Development, with Structural and Mutational Studies Revealing Its DNA-binding Surface

“…S3). These data were consistent with the electrostatic surface most favorable for VRN1(208 -341)-DNA binding and the DNA-binding surface previously identified for the RAV1 B3 domain (9).…”

“…5). Trp-245, Asn-246, Ser-247, and Gln-249 of RAV1 are located in a region that separates two highly basic regions; these residues are proposed to interact directly with DNA bases (9). In both VRN1 and At1g16640, these residues are absent: in VRN1, this region is replaced by a broad continuous basic groove, whereas in At1g16640, a less expansive basic groove is apparent.…”

“…between 2.6 and 3.1 Å for 93-96 C␣ atoms. All three proteins are restriction endonucleases in which a DNA-binding effector domain contains a fold very similar to that found in the B3 family (9).…”

“…We generated recombinant protein from a construct of VRN1 comprising residues 208 -341 (VRN1(208 -341), representing one of the two B3 domains) and show that like the full-length protein, this single B3 domain displays sequence-nonspecific DNA binding properties. We determined a high resolution crystal structure for VRN1(208 -341) and compared it with the NMR structures of two B3 domain proteins that have not been assigned in planta functions: RAV1 is a sequence-specific DNA-binding protein (8,9), whereas At1g16640 is reported not to bind DNA (10). We used NMR to identify putative DNA-binding sites, which we tested with a series of 16 mutations in VRN1(208 -341) that included a triple Arg-to-Glu change that abolished in vitro DNA-binding activity.…”