Fused protein domains inhibit DNA binding by LexA.
Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions.
Golemis
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Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
Brent
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic / Q000502:physiology*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012697:Serine Endopeptidases; D014158:Transcription, Genetic; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein