Cloning and characterization of seven human forkhead proteins: binding site specificity and DNA bending.
The forkhead domain is a monomeric DNA binding motif that defines a rapidly growing family of eukaryotic transcriptional regulators. Genetic and biochemical data suggest a central role in embryonic development for genes encoding forkhead proteins. We have used PCR and low stringency hybridization to isolate clones from human cDNA and genomic libraries that represent seven novel forkhead genes, freac-1 to freac-7. The spatial patterns of expression for the seven freac genes range from specific for a single tissue to nearly ubiquitous. The DNA binding specificities of four of the FREAC proteins were determined by selection of binding sites from random sequence oligonucleotides. The binding sites for all four FREAC proteins share a core sequence, RTAAAYA, but differ in the positions flanking the core. Domain swaps between two FREAC proteins identified two subregions within the forkhead domain as responsible for creating differences in DNA binding specificity. Applying a circular permutation assay, we show that binding of FREAC proteins to their cognate sites results in bending of the DNA at an angle of 80-90 degrees.
Department of Molecular Biology, Göteborg University, Sweden.
S
S
Pierrou
M
M
Hellqvist
L
L
Samuelsson
S
S
Enerbäck
P
P
Carlsson
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D051858:Forkhead Transcription Factors; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; D051858:Forkhead Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors