Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomyces spp. contain genes coding for two RNRs, either of which is sufficient for vegetative growth. The class Ia RNR is encoded by the nrdAB genes, and the class II RNR is encoded by nrdJ, which is coexpressed with nrdR. We previously showed that the Streptomyces coelicolor nrdR gene encodes a protein, NrdR, which represses transcription of both sets of RNR genes. NrdR is a member of a highly conserved family of proteins that is confined exclusively to prokaryotes. In this report, we describe a physical and biochemical characterization of the S. coelicolor NrdR protein and show that it is a zinc-ATP/dATP-containing protein that binds to the promoter regions of both Streptomyces RNR operons. The NrdR N terminus contains a zinc ribbon motif that is necessary for binding to the upstream regulatory region of both RNR operons. The latter contains two 16-bp direct repeat sequences, termed NrdR boxes, which are located proximal to, or overlap with, the promoter regions. These experiments support the view that NrdR controls the transcription of RNR genes by binding to the NrdR box sequences. We also show that the central NrdR ATP cone domain binds ATP and dATP and that mutations that abolish ATP/dATP binding significantly reduce DNA binding, suggesting that the ATP cone domain may allosterically regulate NrdR binding. We conclude that NrdR is a widely conserved regulator of RNR genes, binding to specific sequence elements in the promoter region and thereby modulating transcription.Ribonucleotide reductases (RNRs) provide the building blocks for DNA synthesis and repair in all living cells (20). They are essential because they are the only known de novo pathway for the biosynthesis of deoxyribonucleotides, the immediate precursors for DNA synthesis. Three major classes of RNRs are known (8,15,19). Class I RNRs are oxygen-dependent enzymes that occur in eukaryotes, bacteria, and some viruses, and class II RNRs are oxygen-independent enzymes confined to bacteria and archaea, while class III RNRs are found predominantly in anaerobic bacteria. Despite significant differences in structure and in cofactor requirements, all three enzymes share similar catalytic mechanisms creating a protein radical that initiates reduction of ribonucleotides, and all allosterically regulate the balanced formation of the four deoxyribonucleotides (15,19,24).Previously we reported that Streptomyces spp., gram-positive aerobic bacteria that produce a remarkable variety of metabolites and possess a complex life cycle (7, 13), contain class Ia and class II RNRs (5). Either type of RNR is sufficient for vegetative growth (4). In Streptomyces coelicolor, the class Ia and class II RNRs are regulated by coenzyme B12 (adenosylcobalamin) in a reciprocal manner. B12 negatively controls transcription of the class Ia RNR nrdABS genes via a riboswitch mechanism and positively controls the activity of...