Cooperative and allosterically controlled nucleotide binding regulates the DNA binding activity of NrdR.
Ribonucleotide reductases (RNRs) are required for the synthesis of deoxyribonucleoside triphosphates (dNTPs) from ribonucleotides. In Escherichia coli, regulation of RNR expression is co-ordinated with the cell cycle, and involves several regulatory proteins. One of these, NrdR, has recently been shown to regulate all three nrd operons that encode RNR isoenzymes. Repression by NrdR is believed to be stimulated by elevated dNTPs, although there is no direct evidence for this model. Here, we sought to elucidate the mechanism by which NrdR regulates nrd expression according to the abundance of (d)NTPs. We determined that ATP and dATP bind to NrdR in a negatively cooperative fashion, such that neither can fully occupy the protein. Both nucleotides also appear to act as positive heterotropic effectors, since the binding of one stimulates binding of the other. Nucleotide binding stimulates self-association of NrdR, with tri- and diphosphates stimulating oligomerization more effectively than monophosphates. As-prepared NrdR contains (deoxy)nucleoside monophosphates, diphosphates and triphosphates, and its DNA binding activity is inhibited by triphosphates and diphosphates but not by monophosphates. We propose a model in which NrdR selectively binds (deoxy)nucleoside triphosphates, which are hydrolysed to their monophosphate counterparts in order to regulate DNA binding.
Department of Molecular and Cell Biology, The University of Texas at Dallas, 800 W Campbell Road, Richardson, TX, 75080, USA.
Brandon L
BL
McKethan
Stephen
S
Spiro
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006868:Hydrolysis; D008954:Models, Biological; D009876:Operon; D011485:Protein Binding; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D003838:Deoxyadenine Nucleotides; D029968:Escherichia coli Proteins; C523273:NrdR protein, E coli; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases; C026600:2'-deoxyadenosine triphosphate