This document summarizes research on the RNLAB toxin-antitoxin operon from E. coli. The objective was to determine if alternative dimerization is required for activity and inhibition of the HEPN ribonuclease RnlA. Various techniques were used, including PCR, cell-free expression of the RnlB antitoxin, Western blot, in vivo toxicity assays, and electrophoresis. The results showed that RnlB inhibits the endoribonuclease activity of RnlA. The conclusions discuss how understanding these defense mechanisms could help develop new therapies against bacterial infections and further genetic research.

1. ISABELLA ESCOBAR DOMINGUEZ
JUAN JOSE FIGUEROA
Presented by :
Third Semester
Medicine
Univercidad Pontificia Bolivariana

2. Introduction
THE RNLAB TOXIN-ANTITOXIN OPERON FROM
ESCHERICHIA COLI FUNCTIONS AS AN ANTIPHAGE
DEFENSE SYSTEM.

3. Introduction
-Rn1A
-Rn1B

4. Objective
 Determining alternative dimerization is required for activity and
inhibition of the HEPN ribonuclease RnlA

5. PCR
PCR is a technique for "in vitro" synthesis of specific DNA sequences.
It is a simple and very fast way to multiply the DNA present in different
biological samples
is characterized by being a technique of high sensitivity and efficiency and
this generates more reliable results in a short time and easy to analyze

6. Cell-free expression of RnlB antitoxin
Cell-free translation systems are used for protein expression of either in vitro transcribed mRNA or
mRNA isolated from tissues or cells. These systems are used to express single proteins as well as
multiple proteins in high-throughput applications such as display technologies.

7. Western Blot
Western blot is a laboratory technique used to detect a
specific protein in a sample of blood or tissue.
The method involves the use of gel electrophoresis to
separate proteins from the sample.

8. In vivo toxicity
assay
In vitro toxicology consists of using
cells or tissues maintained or grown
in controlled laboratory conditions
to examine the toxic properties of
compounds and mixtures.

9. Electrophoresis
is the motion of dispersed particles relative to a fluid under the influence of a
spatially uniform electric field. Electrophoresis is used in laboratories to separate
macromolecules based on size. The technique applies a negative charge so
proteins move towards a positive charge. Electrophoresis is used extensively in
DNA, RNA and protein analysis.

10. Results of electrophoresis
A: Rn1A Working time- progress.
B: Effect of RnlB on the activity of RnlA endoribonuclease.

11. Results of electrophoresis

12. Results of electrophoresis

13. Results of electrophoresis

14. Discussion
Xia,Y.,
Site-directed mutagenesis of RnlA was carried out by PCR extension or full-plasmid
amplification with partially overlapping primers
Anantharaman,V. in domains in the abortive infection genes (Abi) system and in domains encoded by
genes related to the CRISPR Cas loci
Otsuka,Y The endoribonuclease activity of RnlA is controlled by RnlB. RnlA is also directly
inhibited by the phage antitoxin encoded by the dmd gene
5
Grynberg,M he HEPN domain consists of five -helices, with three of them arranged in an up-and-
down helical bun- dle and two shorter helices on one side (15)

15. Conclusions
1) The understanding of mechanisms of defense against
viruses like the T4 Phages can lead to the development of
new biomedical strategies to create new types of therapies
against bacterial infecctions other than the use of
antibiotics.
2) The dinamics of the protein behaviour and the effects of
its conformational changes is crucial to the understanding
of mechanisms that can be found both in prokaryotic cells
and eukaryotic organisms.
3) The study of Endoribonucleases like the one presented in
the article can help investigators to develop new tools for
genetic manipulation and this could turn into a new path in
the investigation of pathologies like cancer or genetic
disorders