In vitro analysis of a constitutively active mutant form of the Escherichia coli global transcription factor FNR.
The Escherichia coli transcription factor FNR regulates expression of genes required for the metabolic switch between aerobic and anaerobic respiration. In order to investigate how FNR controls transcription of its target operons, DNA binding was examined for both the wild-type (WT) FNR protein and an altered function FNR* protein (DA154) that exhibits enhanced activity in the presence of oxygen both in vivo and in vitro, apparently due to the fact that DA154 is able to dimerize to a greater extent than WT FNR. Electrophoretic mobility shift assays, using a consensus symmetrical FNR target site, revealed that both DA154 and WT FNR gave rise to protein-DNA complexes of indistinguishable electrophoretic mobilities. In addition, an estimate of the molecular weight from the mobility of the DA154-DNA complex indicated that both mutant and WT FNR were dimeric when bound to DNA. Under the same binding conditions, DA154 showed an observed constant of approximately 3 x 10(8) M-1 for the consensus symmetrical target site. In addition, the results of DNA binding competition assays provided evidence that DA154 was a site-specific DNA binding protein, since this mutant protein bound to the consensus symmetrical target site with approximately 40-fold and 250-fold higher affinity than a natural target site from the nar promoter or a non-specific DNA target, respectively. Electrophoretic mobility shift DNA bending assays demonstrated protein-induced DNA bending by both DA154 and WT FNR. In addition, in vitro transcription assays using an FNR-dependent variant of the lac P1 promoter demonstrated levels of transcription activation by DA154 comparable to those observed in vivo. These results provide several new insights into how FNR functions to activate transcription of target genes.
Department of Bacteriology, University of Wisconsin, Madison 53706.
E C
EC
Ziegelhoffer
P J
PJ
Kiley
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D014157:Transcription Factors