A yeast transcription assay defines distinct rel and dorsal DNA recognition sequences.
Recent data have demonstrated that vRel, cRel, Dorsal, and NF-kappa B are members of a larger family of DNA-binding regulatory proteins. Rel proteins interact to form homo- and heterodimers that recognize specific sites on DNA, and it is likely that such protein-protein and protein-DNA interactions contribute to proper regulation of target gene expression by these proteins. Here we describe the use of a yeast transcription activation assay to study binding of three Rel family proteins to their native binding sites. These results show that the vRel and cRel proteins recognize two known NF-kappa B binding sites; the Dorsal protein does not recognize NF-kappa B sites, but does recognize related sites upstream of the Drosophila zerknüllt gene. Our experiments demonstrate that the members of this protein family recognize similar, but not identical, sites in the promoters of target genes, and we are able to identify a particular nucleotide that is apparently involved in the DNA-protein interaction. We exploit the properties of LexA fusion proteins to study the dimerization and DNA-contacting domains of cRel. Our results suggest that the cRel protein forms homodimers and that dimer formation may be necessary for cRel to bind DNA. Finally, our results show that transcription activation by these proteins is cooperative; such cooperativity may be important for correct temporal and spatial regulation of target gene expression.
Kamens
J
J
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
Brent
R
R
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D001681:Biological Assay / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005813:Genes, Synthetic; D005821:Genetic Techniques; D007301:Insect Hormones / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D016328:NF-kappa B / Q000378:metabolism; D020572:Oncogene Proteins v-rel; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D020569:Proto-Oncogene Proteins c-rel; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D007301:Insect Hormones; D016328:NF-kappa B; D020572:Oncogene Proteins v-rel; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015682:Retroviridae Proteins, Oncogenic; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein; C052130:GAL4 protein, S cerevisiae