Investigation of mRNA Recognition and Specificity by the Type II Toxin YoeB in Escherichia coli Restricted; Files Only

Abstract

Toxin-antitoxin (TA) modules are gene pair loci ubiquitous in bacterial cells that encode for a toxin, inhibitors of cell growth, and its cognate antitoxin which counteracts the toxin. In general, toxins are proteins and inhibit cell growth by targeting crucial processes such as DNA replication or protein translation, whereas the antitoxin can either be a protein or RNA. Currently, there are eight known types of TA modules, classified based on the nature of the antitoxin and its mode of repression of the toxin. The best studied TA systems are the type II TA modules; the main characteristic of these is that both toxin and antitoxin are proteins. Under normal physiological conditions, the antitoxin is directly bound to the toxin, inhibiting toxin activity. The addition of stress to the system releases the labile antitoxin from the toxin, resuming toxin activity. My research investigates the type II toxin YoeB, a ribosome-dependent endoribonuclease that targets mRNA actively involved in translation. YoeB selectively cleaves mRNA at the ribosomal aminoacyl (A) site between the second and third nucleotide and confers specificity for certain nucleotide sequences. This paper investigates the specificity and recognition mechanisms of YoeB to its mRNA substrates by generating variants of the YoeB protein. Through the systematic substitution of the YoeB Glu62 residue, we may better understand how this toxin targets mRNA and define how specificity for cleavage relates to its biological function. 

Table of Contents

Table of Contents

Introduction

Type II Toxin-Antitoxin Modules....................................................................................... 1

Ribosome-Dependent Type II TA Modules.......................................................................... 2

Defining mRNA Specificity in Ribosome-Dependent Toxins................................................. 4

Investigation of YoeB Glu62.............................................................................................. 6

Significance: Antimicrobial Resistance and Bacterial Persistence......................................... 7

Materials and Methods

Strains and Plasmids......................................................................................................... 9

Primer Design and Site-directed Mutagenesis..................................................................... 9

Induction of YefMYoeB, YoeB, and YoeB variants................................................................. 11

Spot Dilution Assays......................................................................................................... 11

Bacterial Growth Assays.................................................................................................... 12

Results and Discussion

Spot Dilutions of YoeB Variants......................................................................................... 14

Growth Curves of YoeB Variants........................................................................................ 15

Small-scale Induction of YoeB E62A and E62D for Protein Purification................................. 17

Conclusion

Effects of YoeB Variants on In vivo Function....................................................................... 18

Future Directions............................................................................................................. 18

References....................................................................................................................... 20

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry
Stichwort	ribosome toxin-antitoxin modules biochemistry endoribonuclease
Committee Chair / Thesis Advisor	Dunham, Christine, Emory University
Committee Members	Conticello, Vincent, Emory University Nkomo, Simbarashe, Emory University

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