The Streptomyces NrdR Transcriptional Regulator Is a Zn Ribbon/ATP Cone Protein That Binds to the Promoter Regions of Class Ia and Class II Ribonucleotide Reductase Operons

Abstract

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...

Functional Analysis of the Streptomyces coelicolor NrdR ATP-Cone Domain: Role in Nucleotide Binding, Oligomerization, and DNA Interactions

“…The ATP-cone domain alone determines nucleotide binding since a truncated protein that contains only that domain binds ATP/dATP (18). Moreover, a NrdR ATP-cone mutant that is defective in nucleotide binding was found to be unable to bind short DNA probes containing NrdR-boxes (18). These observations led us to propose that when NrdR binds ATP/dATP it undergoes a conformational change that facilitates binding to its cognate DNA recognition sequences to repress RNR gene expression.…”

“…Single point mutations in the ATPcone domain-Val483Ala, Lys503Ala, Arg513Ala, Glu563Ala, Lys623Ala, Val633Ala, Tyr1213Ala, and Tyr1283Ala-were created by an overlap PCR procedure as described previously (18). Two PCR fragments were amplified from M145 genomic DNA by use of two nonmutagenic external oligonucleotides, the forward primer IG-1 (5Ј-ATATCATATGCACTGCCCCTTTGC-3Ј) contains an NdeI restriction site and the reverse primer IG-2 (5Ј-TCTCAAGCTTGTCGG CGGCGCCTGCGG-3Ј) contains a HindIII restriction site (restriction sites are underlined), as well as two complementary mutagenic internal oligonucleotides ( Table 1).…”

“…S. coelicolor NrdR contains up to one mole of tightly bound ATP or dATP per mole protein. The ATP-cone domain alone determines nucleotide binding since a truncated protein that contains only that domain binds ATP/dATP (18). Moreover, a NrdR ATP-cone mutant that is defective in nucleotide binding was found to be unable to bind short DNA probes containing NrdR-boxes (18).…”

“…Immediately following, an ϳ90-amino-acid sequence is predicted to form an ATP-cone domain similar to that present in the overall effector activity site of E. coli NrdA (5). We previously showed that an intact zinc ribbon domain is necessary for binding of NrdR to conserved tandem 16-bp sequences, termed NrdR-boxes, located in the upstream regulatory regions of both S. coelicolor RNR operons (18). Rodionov and Gelfand (38) subsequently used phylogenetic profiling to show that the location of NrdR-boxes is almost invariably correlated with that of RNR operons.…”

Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays

“…Structure and function predictions for this protein family indicate the presence of an amino-terminal zinc b-ribbon domain and an ATP-cone domain that shows similarity to the allosteric effector domain of certain ribonucleotide reductases (Aravind et al, 2000;Rodionov & Gelfand, 2005). NrdR proteins are involved in transcriptional regulation of ribonucleotide reductase gene expression in bacteria by binding to operators termed NrdR boxes (Grinberg et al, 2006;Rodionov & Gelfand, 2005;Torrents et al, 2007). Conserved NrdR boxes are located in front of the nrdHIE genes of C. glutamicum, which encode subunits of a class Ib ribonucleotide reductase (Rodionov & Gelfand, 2005).…”