Escherichia coli possesses class Ia, class Ib, and class III ribonucleotide reductases (RNR). Under standard laboratory conditions, the aerobic class Ia nrdAB RNR genes are well expressed, whereas the aerobic class Ib nrdEF RNR genes are poorly expressed. The class III RNR is normally expressed under microaerophilic and anaerobic conditions. In this paper, we show that the E. coli YbaD protein differentially regulates the expression of the three sets of genes. YbaD is a homolog of the Streptomyces NrdR protein. It is not essential for growth and has been renamed NrdR. Previously, Streptomyces NrdR was shown to transcriptionally regulate RNR genes by binding to specific 16-bp sequence motifs, NrdR boxes, located in the regulatory regions of its RNR operons. All three E. coli RNR operons contain two such NrdR box motifs positioned in their regulatory regions. The NrdR boxes are located near to or overlap with the promoter elements. DNA binding experiments showed that NrdR binds to each of the upstream regulatory regions. We constructed deletions in nrdR (ybaD) and showed that they caused high-level induction of transcription of the class Ib RNR genes but had a much smaller effect on induction of transcription of the class Ia and class III RNR genes. We propose a model for differential regulation of the RNR genes based on binding of NrdR to the regulatory regions. The model assumes that differences in the positions of the NrdR binding sites, and in the sequences of the motifs themselves, determine the extent to which NrdR represses the transcription of each RNR operon.Ribonucleotide reductases (RNRs) are essential enzymes in all living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides, the immediate precursors of DNA synthesis and repair (34). Three major classes of RNRs have been characterized. Class I RNRs are oxygendependent enzymes that occur in eubacteria, eukaryotes, and some viruses; class II RNRs are oxygen-independent enzymes confined to bacteria, archaea, and a few unicellular eukaryotes; and class III RNRs are oxygen-sensitive enzymes present in anaerobes. Despite significant differences in their structures and in cofactor requirements, all three enzymes share similar catalytic mechanisms creating a transient cysteinyl radical that initiates the reduction of ribonucleotides, and all employ sophisticated allosteric mechanisms that enable the balanced formation of each of the four deoxyribonucleotides (13, 34).While eukaryotes in general employ just the class I RNR, many bacteria possess two or even all three RNR classes, the genes of which are typically organized in operons (45) (for a comprehensive listing of RNRs in bacteria, see http://rnrdb .molbio.su.se). Escherichia coli, Salmonella enterica serovar Typhimurium, and many enterobacteria contain genes encoding three RNRs, class Ia and class Ib RNRs (subdivisions of the class I RNR) and the class III RNR. In E. coli, the class Ia RNR operon contains nrdA and nrdB genes that code for the NrdA catalytic and the ...