Samenvatting
The staggering biodiversity of microbes stands as a testament to the long evolutionary history of the oldest lifeforms on Earth. We are only now beginning to unravel, as technological breakthroughs open new horizons for science, the magnificent molecular machineries that constitute the basis of microbial structures and hence function. Horizontal gene transfer events in prokaryotic cells, as well as the arms race between bacteriophages and their hosts, assisted in the wide spread of molecular weapons that target many vital cell processes and their counteracting entities that provide a natural defense. These prokaryotic toxins are ubiquitous and abundant in the genomes of almost all species, and yet they exist under tight regulation to protect the cell against self-harm under homeostasis. The molecular mechanisms by which prokaryotes accomplish this task take place at many levels of regulation, i.e., direct inhibition of protein activity by protein-protein interactions, transcriptional regulation by repressor binding to DNA promoter to abrogate the activity of RNA polymerase, etc. By looking at the molecular level into the activity and regulation of the Escherichia coli RnlA-RnlB toxin-antitoxin (TA) system I have unveiled a novel regulatory mechanism exerted on the toxin RnlA by its cognate chromosomic antitoxin RnlB. This toxin functions as an endoribonuclease first reported as the host-encoded RNase responsible for the T4 phage late gene silencing when a dmd- mutant of the phage infects the cells, halting its propagation. Here I show that RnlA is a HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain) ribonuclease with broad sequence specificity in vitro. Comparative bioinformatics led to the identification of the catalytic residues in RnlA, which upon mutation into alanine residues permitted the mapping of the active site of this enzyme. The drastic conformational changes on the structure of RnlA exerted by its antitoxin RnlB and detected by X-ray crystallography and SAXS, constitute the basis for its inhibition and the first example of a mechanism involving quaternary structural changes halting the activity of a ribonuclease known to the scientific community.The toxic entities can also exert its function by directly binding to and blocking essential targets in the cell, like topoisomerases, key players in maintaining the right balance of DNA supercoiling, which is essential for cell growth and division. ParE2 is a gyrase poison encoded in chromosome II of the human pathogen Vibrio cholerae, which is directly counteracted by its cognate antitoxin ParD2. Here I have determined the crystallographic structure of the ParD2-ParE2 complex, which unveiled the molecular basis for the control of this toxin's poisonous effect by direct interaction with its cognate antitoxin. Moreover, I have also mapped the operator region upstream of the parde2 operon where this TA system functions as its own transcriptional repressor, as well as determined the stoichiometry of the ParD2-ParE2 complex by X-ray crystallography, SAXS and native mass spectrometry studies. Electrophoretic mobility shift assays point to the presence of conditional cooperativity in the transcriptional autoregulation of this system.
| Datum prijs | 5 okt 2020 |
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| Originele taal | English |
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