Biology subjects

Rozman Grinberg, I.

Publications and source records attributed to Rozman Grinberg, I..

5 recordsLinked to original sources

bioRxiv2026

Mutational and bioinformatic analysis of the binding site for the ribonucleotide reductase-specific transcriptional repressor NrdR

The prevalent transcriptional repressor NrdR binds to highly conserved prokaryotic sequences in the promoter regions of operons encoding the essential enzyme ribonucleotide reductase. The NrdR binding sites consist of two partially palindromic 16 bp sequences (NrdR boxes) separated by a 15-16 bp linker sequence. We have assessed the requirement of both boxes for binding, the propensity of different NrdRs to bind to heterologous binding sites, and that the linker sequence is only limited to length and not sequence conservation. As we have observed several deviations from the conserved sequences of the NrdR boxes, we here test the conservation requirements of individual basepairs in the NrdR boxes using a synthetic DNA fragment (Synt DNA) to which the NrdR proteins from the actinomycete Streptomyces coelicolor and the gammaproteobacterium Escherichia coli bind equally well as to their homologous binding sites. By introducing isolated mutations to Synt DNA and testing the binding capacity of NrdR from S. coelicolor and E. coli we expand our understanding of what criteria are needed to build a functional binding site for the NrdR repressor.

molecular biology↗

bioRxiv2024

Bacterial transcriptional repressor NrdR - a flexiblemultifactorial nucleotide sensor

NrdR is a bacterial transcriptional repressor consisting of a Zn-ribbon domain followed by an ATP-cone domain. Understanding its mechanism of action could aid the design of novel antibacterials. NrdR binds specifically to two "NrdR boxes" upstream of ribonucleotide reductase operons, of which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, where we identified a new box. We show that E. coli NrdR (EcoNrdR) has similar binding strength to all three sites when loaded with ATP plus dATP or equivalent diphosphate combinations. No other combination of nucleotides promotes binding to DNA. We present crystal structures of EcoNrdR-ATP-dATP and EcoNrdR-ADP-dATP, which are the first high-resolution crystal structures of an NrdR. We have also determined cryo-EM structures of DNA-bound EcoNrdR-ATP-dATP and novel filaments of EcoNrdR-ATP. Tetrameric forms of EcoNrdR involve alternating interactions between pairs of Zn-ribbon domains and ATP-cones. The structures reveal considerable flexibility in relative orientation of ATP-cones vs Zn-ribbon domains. The structure of DNA-bound EcoNrdR-ATP-dATP shows that significant conformational rearrangements between ATP-cones and Zn-ribbons accompany DNA binding while the ATP-cones retain the same relative orientation. In contrast, ATP-loaded EcoNrdR filaments show rearrangements of the ATP-cone pairs and sequester the DNA-binding residues of NrdR such that they are unable to bind to DNA. Our results, in combination with a previous structural and biochemical study, point to highly flexible EcoNrdR structures that when loaded with the correct nucleotides adapt to an optimal promoter binding conformation.

biophysics↗

bioRxiv2024

Strong conservation of spacer lengths in NrdR repressor DNA binding sites

The ribonucleotide reductase-specific repressor NrdR, from the human pathogens Listeria monocytogenes and Streptococcus pneumoniae, form tetramers that bind to DNA when loaded with dATP and ATP. If loaded with only ATP they form different oligomeric complexes that cannot bind to DNA. The DNA binding site in L. monocytogenes is a pair of NrdR boxes separated by 15-16 bp, whereas in Streptococcus pneumoniae the NrdR boxes are separated by 25-26 bp. However, Streptococcus pneumoniae NrdR binds stronger to the related Streptococcus thermophilus binding sites with NrdR boxes separated by 15-16 bp. This observation triggered a comprehensive binding study of four NrdRs from L. monocytogenes, Streptococcus pneumoniae, Escherichia coli and Streptomyces coelicolor to a series of synthetic dsDNA fragments where the NrdR boxes were separated by 12-27 bp. All four NrdRs bound well to NrdR boxes separated by 14-17 bp, and also to NrdR boxes separated by 24-27 bp. The worst binding occurred when NrdR boxes were separated by 20 bp. The in vitro results were confirmed in vivo in E. coli for spacer distances 12-27 bp. We conclude that NrdR repressors bind most efficiently when there is an integer number of DNA turns between the center of the two NrdR boxes.

biochemistry↗

bioRxiv2018

Fusion of a functional glutaredoxin to the radical-generating subunit of ribonucleotide reductase

Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radical-generating subunit (NrdB) that together catalyse reduction of the four ribonucleotides to their corresponding deoxyribonucleotides. Facklamia ignava NrdB is an unprecedented fusion protein with N-terminal add-ons of a glutaredoxin (Grx) domain followed by an ATP-cone. Grx, which in general is encoded elsewhere in the genome than is the RNR operon, is a known physiological reductant of RNRs. Here we show that the fused Grx domain functions as an efficient reductant of the F. ignava class I RNR via the common dithiol mechanism and interestingly also via a monothiol mechanism, although less efficiently. A Grx that utilizes either or of these two reaction mechanisms has to our knowledge not been observed with a native substrate before. The ATP-cone, which is commonly found as an N-terminal domain of the catalytic subunit of RNRs, is an allosteric on/off switch that promotes dNDP reduction in presence of ATP and inhibits the enzyme activity in presence of dATP. Here we show that dATP bound to the ATP-cone of F. ignava NrdB promotes formation of tetramers that are unable to form enzymatically competent complexes with F. ignava NrdA. The ATP-cone binds two molecules of dATP, but only one molecule of the activating nucleotide ATP. F. ignava NrdB contains the recently identified radical factor Mn2III/IV. We show that NrdA from the firmicute F. ignava can form a catalytically competent RNR with the Mn2III/IV-containing NrdB from the flavobacterium Leeuwenhoekiella blandensis.

biochemistry↗

bioRxiv2017

Unique ATP-cone-driven allosteric regulation of ribonucleotide reductase via the radical-generating subunit

Ribonucleotide reductases (RNRs) are key enzymes in DNA synthesis and repair, with sophisticated allosteric mechanisms controlling both substrate specificity and overall activity. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the Leewenhoekiella blandensis radical-generating subunit regulates activity via modifications of quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzyme complexes. The tetramer forms solely by interactions between ATP-cones, as evidenced by a 2.45 [A] crystal structure. We also present evidence for an MnIIIMnIV metal center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated by evolutionary capture of the domain by the radical-generating subunit. Our findings present a novel opportunity for dATP-regulation of engineered proteins.

biochemistry↗