Analysis of the NF-kappaB p50 dimer interface by diversity screening.
An in vivo screen has been devised for NF-kappaB p50 activity in Escherichia coli exploiting the ability of the mammalian transcription factor to emulate a prokaryotic repressor. Active intracellular p50 was shown to repress the expression of a green fluorescent protein reporter gene allowing for visual screening of colonies expressing active p50 on agar plates. A library of mutants was constructed in which the residues Y267, L269, A308 and V310 of the dimer interface were simultaneously randomised and twenty-five novel functional interfaces were selected which repressed the reporter gene to similar levels as the wild-type protein. The leucine-269 alanine-308 core was repeatedly, but not exclusively, selected from the library whilst a diversity of predominantly non-polar residues were selected at positions 267 and 310. These results indicate that L269 and A308 may form a hot spot of interaction and allow an insight into the processes of dimer selectivity and evolution within this family of transcription factors.
Hart
D J
DJ
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK.
Speight
R E
RE
Sutherland
J D
JD
Blackburn
J M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D019143:Evolution, Molecular; D017930:Genes, Reporter / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D016328:NF-kappa B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D020569:Proto-Oncogene Proteins c-rel / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics; D014169:Transformation, Bacterial
D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D020569:Proto-Oncogene Proteins c-rel; D012097:Repressor Proteins; D012269:Ribosomal Proteins; C016700:ribosomal protein L7-L12; D004247:DNA