Structure and function of multicomponent complexes
[eng] The emergence and spread of antibiotic resistance among bacteria has become an undisputed global problem and one of the greatest threats to public health in the 21st century. The widespread, excessive and uncontrolled use of antibiotics, not only for therapeutic purposes but also in agricultur...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2023 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/199423 |
| Acceso en línea: | https://hdl.handle.net/2445/199423 http://hdl.handle.net/10803/688475 |
| Access Level: | acceso abierto |
| Palabra clave: | Factors de transcripció Proteïnes Oligòmers Mutagènesi Transcription factors Proteins Oligomers Mutagenesis |
| Sumario: | [eng] The emergence and spread of antibiotic resistance among bacteria has become an undisputed global problem and one of the greatest threats to public health in the 21st century. The widespread, excessive and uncontrolled use of antibiotics, not only for therapeutic purposes but also in agriculture and animal husbandry, has resulted in a steady and rapid increase in the number of strains resistant to the drugs used. In all living cells, ribonucleotide reductases (RNRs) are essential enzymes that constitute the only known de novo pathway of deoxyribonucleotide biosynthesis (dNTP biosynthesis) via the catalyzed reduction of ribonucleoside triphosphates (NTPs, such as ATP, CTP, GTP, and TTP) using radical chemistry, thereby forming the fundamental building blocks for DNA synthesis and repair. The transcriptional repressor NrdR controls the expression of RNR genes in most bacteria and in some archaea. Importantly, NrdR is missing in eukaryotes, and as it is found in antibiotic resistant pathogens such as Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Staphylococcus aureus, it can be considered as a biomedical target. NrdR inhibits transcription of RNRs genes by binding its Zn-finger domain (ZFD) to a palindromic repeat of 16bp DNA that conform the so-called NrdR-boxes, which are upstream of RNRs promoter regions. An ATP-cone domain (ACD) present in NrdR, sensitive to the changes in concentration of (-deoxy) ribonucleoside triphosphates (dNTPs and NTPs), allosterically regulates the Zn-finger activity of NrdR. Several hypotheses on RNRs genes regulation by NrdR have been proposed, and in May 2022 a combination of biochemical and cryo-EM structural studies suggested mechanism of action for Streptomyces coelicolor (sc-) NrdR. Such a mechanism involves an ATP-loaded dodecamer, which cannot bind to DNA, a dATP/ATP- loaded octamer, and a dATP/ATP-loaded tetramer bound to the sc-nrdRJ promoter, which represses transcription of the RNR operon. In this doctoral thesis, the crystal structure of Escherichia coli NrdR revealed key interactions whose mutations altered the multimerization. To test the functional roles played by the different residues, in vitro assays were carried out that showed in solution the WT NrdR dimer instability in the absence of nucleotides, and elution of different assemblies in the presence of AMP, ADP, ATP, and dATP. The same studies performed with fusion NrdR and designed mutants in which the multimerization interactions were altered, revealed the importance of residues Glu36, Glu42, Tyr131 at the ZFD, and of segment aa 132-149 from the ACD in NrdR oligomerization. The highest impact was noted for mutation Glu42Ala and the deletion of aa 132-149 segment. Thus, both the ZFD and the ACD are fundamental for oligomerization and essential for the protein function. In vivo studies performed with single-site mutants indicated that mutation E42A at the ZFD completely abolished NrdR ability to repress transcription of RNRs, while mutation to Ala of multimerization-sensitive residues Glu36 at the ZFD and Tyr131 at the ACD did not cause a decrease of the repression level. Therefore, amino acid Glu42 is pivotal for the repressive function of RNR. The abundance of the NrdR protein in bacteria and extrapolation of the results obtained for E. coli and S. coelicolor NrdR points to an ATP/dATP-orchestrated mechanism, in which the type of NrdR multimers change and coordinates the repression activity of the RNR operon. |
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