Common and variable contributions of Fis residues to high-affinity binding at different DNA sequences.
Fis is a nucleoid-associated protein that interacts with poorly related DNA sequences with a high degree of specificity. A difference of more than 3 orders of magnitude in apparent Kd values was observed between specific (Kd, approximately 1 to 4 nM) and nonspecific (Kd, approximately 4 microM) DNA binding. To examine the contributions of Fis residues to the high-affinity binding at different DNA sequences, 13 alanine substitutions were generated in or near the Fis helix-turn-helix DNA binding motif, and the resulting proteins were purified. In vitro binding assays at three different Fis sites (fis P II, hin distal, and lambda attR) revealed that R85, T87, R89, K90, and K91 played major roles in high-affinity DNA binding and that R85, T87, and K90 were consistently vital for binding to all three sites. Other residues made variable contributions to binding, depending on the binding site. N84 was required only for binding to the lambda attR Fis site, and the role of R89 was dramatically altered by the lambda attR DNA flanking sequence. The effects of Fis mutations on fis P II or hin distal site binding in vitro generally correlated with their abilities to mediate fis P repression or DNA inversion in vivo, demonstrating that the in vitro DNA-binding effects are relevant in vivo. The results suggest that while Fis is able to recognize a minimal common set of DNA sequence determinants at different binding sites, it is also equipped with a number of residues that contribute to the binding strength, some of which play variable roles.
Feldman-Cohen
Leah S
LS
Department of Chemistry, College of Staten Island and Macromolecular Assemblies Institute of the City, University of New York, Staten Island 10314, USA.
Shao
Yongping
Y
Meinhold
Derrick
D
Miller
Charmi
C
Colón
Wilfredo
W
Osuna
Robert
R
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001287:Attachment Sites, Microbiological; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D035021:Factor For Inversion Stimulation Protein; D015964:Gene Expression Regulation, Bacterial; D019077:Helix-Turn-Helix Motifs / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding; D011995:Recombination, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; C501034:Fis protein, E coli; D014157:Transcription Factors