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RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants.

ABSTRACT: We have cloned and characterized two novel DNA binding proteins designated RAV1 and RAV2 from Arabidopsis thaliana. RAV1 and RAV2 contain two distinct amino acid sequence domains found only in higher plant species. The N-terminal regions of RAV1 and RAV2 are homologous to the AP2 DNA-binding domain present in a family of transcription factors represented by the Arabidopsis APETALA2 and tobacco EREBP proteins, while the C-terminal region exhibits homology to the highly conserved C-terminal domain, designated B3, of VP1/ABI3 transcription factors. Binding site selection assays using a recombinant glutathione S-transferase fusion protein have revealed that RAV1 binds specifically to bipartite recognition sequences composed of two unrelated motifs, 5'-CAACA-3' and 5'-CACCTG-3', separated by various spacings in two different relative orientations. Analyses using various deletion derivatives of the RAV1 fusion protein show that the AP2 and B3-like domains of RAV1 bind autonomously to the CAACA and CACCTG motifs, respectively, and together achieve a high affinity and specificity of binding. From these results, we suggest that the AP2 and B3-like domains of RAV1 are connected by a highly flexible structure enabling the two domains to bind to the CAACA and CACCTG motifs in various spacings and orientations.

SUBMITTER: Kagaya Y

PROVIDER: S-EPMC148202 | biostudies-other | 1999 Jan

REPOSITORIES: biostudies-other

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RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants.

Kagaya Y Y Ohmiya K K Hattori T T

Nucleic acids research 19990101 2

We have cloned and characterized two novel DNA binding proteins designated RAV1 and RAV2 from Arabidopsis thaliana. RAV1 and RAV2 contain two distinct amino acid sequence domains found only in higher plant species. The N-terminal regions of RAV1 and RAV2 are homologous to the AP2 DNA-binding domain present in a family of transcription factors represented by the Arabidopsis APETALA2 and tobacco EREBP proteins, while the C-terminal region exhibits homology to the highly conserved C-terminal domain ...[more]

PMID: 9862967

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Project description:The REB1 gene encodes a DNA-binding protein (Reb1p) that is essential for growth of the yeast Saccharomyces cerevisiae. Reb1p binds to sites within transcriptional control regions of genes transcribed by either RNA polymerase I or RNA polymerase II. The sequence of REB1 predicts a protein of 809 amino acids. To define the DNA-binding domain of Reb1p, a series of 5' and 3' deletions within the coding region was constructed in a bacterial expression vector. Analysis of the truncated Reb1p proteins revealed that nearly 400 amino acids of the C-terminal portion of the protein are required for maximal DNA-binding activity. To further define the important structural features of Reb1p, the REB1 homolog from a related yeast, Kluyveromyces lactis, was cloned by genetic complementation. The K. lactis REB1 gene supports active growth of an S. cerevisiae strain whose REB1 gene has been deleted. The Reb1p proteins of the two organisms generate almost identical footprints on DNA, yet the K. lactis REB1 gene encodes a polypeptide of only 595 amino acids. Comparison of the two Reb1p sequences revealed that within the region necessary for the binding of Reb1p to DNA were two long regions of nearly perfect identity, separated in the S. cerevisiae Reb1p by nearly 150 amino acids but in the K. lactis Reb1p by only 40 amino acids. The first includes a 105-amino-acid region related to the DNA-binding domain of the myb oncoprotein; the second bears a faint resemblance to myb. The hypothesis that the DNA-binding domain of Reb1p is formed from these two conserved regions was confirmed by deletion of as many as 90 amino acids between them, with little effect on the DNA-binding ability of the resultant protein. We suggest that the DNA-binding domain of Reb1p is made up of two myb-like regions that, unlike myb itself, are separated by as many as 150 amino acids. Since Reb1p protects only 15 to 20 nucleotides in a chemical or enzymatic footprint assay, the protein must fold such that the two components of the binding site are adjacent.

Dataset Information

RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants.

Publications

RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants.

Similar Datasets

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