Interaction between proteins containing homeodomains associated to leucine zippers from sunflower.
A strategy based on the use of PCR with one degenerate oligonucleotide deduced from conserved sequences and lambda gt10 primers was used to isolate homeobox containing sequences from sunflower stem and root cDNA libraries. Six different partial cDNAs coding for the first 48 amino acids of homeodomains and amino terminal sequences were analyzed and found to be members of the HD-Zip superfamily, which contain a homeobox linked to a leucine zipper coding region. A full-length cDNA clone, Hahb-10, was isolated and characterized. The leucine zipper portions of Hahb-10 and of the previously reported Hahb-1 have been utilized to construct fusions with the N-terminal domain of the lambda repressor. These fusions were tested for their ability to bind to lambda promoters in vivo. The expression of a protein containing an active dimerization domain, but not capable of DNA binding, exerts a dominant negative effect on the ability of repressor-zipper fusions to bind to its target DNA. From these experiments, it was concluded that Hahb-1 and -10, when co-expressed, form preferentially homodimers. Exchange of conserved threonines and leucines at positions a1 and d1 of both zippers reduces dimerization efficiency and allows the formation of heterodimers, suggesting that these residues are, among others, determinants of the specificity of interaction, most likely through changes in hydrophobic packing interactions at the dimer interface. The results imply that a great number of interacting molecular entities compose this protein superfamily which is presumably involved in regulating plant developmental responses.
Gonzalez
D H
DH
Area Biología Molecular, Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Rosario, Argentina. dhgon@unrobi.edu.ar
Valle
E M
EM
Chan
G G
GG
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D015723:Gene Library; D017343:Genes, Plant; D006368:Helianthus / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D016350:Leucine Zippers / Q000502:physiology*; D008969:Molecular Sequence Data; D005810:Multigene Family; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D018076:DNA, Complementary; C091850:HD-Zip protein, Helianthus annuus; D018398:Homeodomain Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins