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Regulatory mechanisms behind the activities of bacterial HEPN ribonuclease RnlA and ParE2 gyrase poison

Book - Dissertation

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.
Publication year:2020
Keywords:Molecular Biophysics
  • ORCID: /0000-0002-7996-7622/work/109916385