A nucleotide-sensing oligomerization mechanism that controls NrdR-dependent transcription of ribonucleotide reductases.

Ribonucleotide reductase (RNR) is an essential enzyme that catalyzes the synthesis of DNA building blocks in virtually all living cells. NrdR, an RNR-specific repressor, controls the transcription of RNR genes and, often, its own, in most bacteria and some archaea. NrdR senses the concentration of nucleotides through its ATP-cone, an evolutionarily mobile domain that also regulates the enzymatic activity of many RNRs, while a Zn-ribbon domain mediates binding to NrdR boxes upstream of and overlapping the transcription start site of RNR genes. Here, we combine biochemical and cryo-EM studies of NrdR from Streptomyces coelicolor to show, at atomic resolution, how NrdR binds to DNA. The suggested mechanism involves an initial dodecamer loaded with two ATP molecules that cannot bind to DNA. When dATP concentrations increase, an octamer forms that is loaded with one molecule each of dATP and ATP per monomer. A tetramer derived from this octamer then binds to DNA and represses transcription of RNR. In many bacteria - including well-known pathogens such as Mycobacterium tuberculosis - NrdR simultaneously controls multiple RNRs and hence DNA synthesis, making it an excellent target for novel antibiotics development.

Genetic Variability and Population Structure of the Tunisian Sicilo-Sarde Dairy Sheep Breed Inferred from Microsatellites Analysis.

This study analyzed the genetic variability, inbreeding and population structure of the Tunisian-North African dairy sheep breed, the Sicilo-Sarde (SS), created by crossing the Sarda and Comisana dairy breeds. The level of variability in the SS, considered as an endangered breed after a dramatic decrease, was assessed using 17 microsatellite markers by analyzing the two breed populations sampled from their respective cradles: SS of Beja (SSB, n = 27) and SS of Mateur (SSM, n = 25). High levels of genetic diversity in SS were revealed, with a total of 212 alleles, a high mean number of alleles (12.47 ± 4.17) and a high average polymorphism information content (PIC) (0.81 ± 0.10). The observed heterozygosity was considerable in SSB and SSM (0.795 and 0.785, respectively). The inbreeding level measured by the population inbreeding coefficient FIS is higher in the SSM population (0.121) than in the SSB population (0.090). The higher genetic diversity level detected in SSB reflected the effect of new Italian Sarda genes introduced by intra-uterine artificial insemination recently practiced in this population. The Wilcoxon test and the mode-shift distribution indicated that the SS breed is a non-bottlenecked population. The structural analysis reflected the historical miscegenation practiced during the breed creation and highlighted further ancient miscegenation, which could date back to the first waves of sheep introduction to the western Mediterranean region. Microsatellite markers were successfully applied in the assessment of the genetic variability of SS and should be used in monitoring this variability during the application of conservation strategies.

Structural determinants and distribution of phosphate specificity in ribonucleotide reductases.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the building blocks of DNA. RNRs are specific for either ribonucleoside diphosphates or triphosphates as substrates. As far as is known, oxygen-dependent class I RNRs (NrdAB) all reduce ribonucleoside diphosphates, and oxygen-sensitive class III RNRs (NrdD) are all ribonucleoside triphosphate reducers, whereas the adenosylcobalamin-dependent class II (NrdJ) contains both ribonucleoside diphosphate and triphosphate reducers. However, it is unknown how this specificity is conveyed by the active site of the enzymes and how this feature developed in RNR evolution. By structural comparison of the active sites in different RNRs, we identified the apical loop of the phosphate-binding site as a potential structural determinant of substrate specificity. Grafting two residues from this loop from a diphosphate- to a triphosphate-specific RNR caused a change in preference from ribonucleoside triphosphate to diphosphate substrates in a class II model enzyme, confirming them as the structural determinants of phosphate specificity. The investigation of the phylogenetic distribution of this motif in class II RNRs yielded a likely monophyletic clade with the diphosphate-defining motif. This indicates a single evolutionary-split event early in NrdJ evolution in which diphosphate specificity developed from the earlier triphosphate specificity. For those interesting cases where organisms contain more than one nrdJ gene, we observed a preference for encoding enzymes with diverse phosphate specificities, suggesting that this varying phosphate specificity confers a selective advantage.