Part:BBa_K2142003

mazF toxin

This sequence encodes for the mazF protein. This protein is the toxin for the mazE antitoxin (BBa_K2142001). Together the parts form the mazF/mazE system which can be used as selectable marker. This part is not a standard BioBrick and can be found in BBa_K2142005 Sequence and Features.

Introduction

Vilnius-Lithuania iGEM 2020 project FlavoFlowincludes three goals towards looking for Flavobacterium disease-related problems’ solutions. The project includes creating a rapid detection kit, based on HDA and LFA, developing an implement for treating a disease, and introducing the foundation of edible vaccines. This part was used for the second goal- treatment - of the project FlavoFlow.

Biology

Toxin-antitoxin (TA) system is abundant in all bacteria. The TA presence in bacteria is as a response to stress conditions¹. The comprehensively investigated system is mazEF in Escherichia Coli ^2,3. These kinds of systems regulate cell death and survival. During cell growth, the toxin is cohered with antitoxin⁴. The antitoxin degrades and detaches from a toxin in the presence of stress conditions. The mazEF system belongs to type II toxin and antitoxin interaction. The most common toxins are ribonucleases in type II TA⁵.

MazF is a toxin in the toxin-antitoxin complex. MazF – sequence-specific endoribonuclease, which cleaves mRNA at ACA site and terminates protein synthesis⁶. The ACA site is present in 96% of E.Coli coding sequences. MazF is a stable protein that is inhibited by MazE(K2142001). The target list complements and the 16S rRNA of the 30S ribosomal subunit. MazF cleaves at an ACA triplet in the 16S rRNA located 5‘ of helix 45. It is lost about 43 nucleotides at the 3‘ C terminus of 16S rRNA, including 45 helix and the anti-Shine-Dalgarano (aSD) sequence during this cleavage⁷. The SD-aSD interaction is necessary for protein synthesis initiation. The MazF is the toxic element in the mazEF heterocomplex. This module exists as a polypeptide chain which includes one globular MazE homodimer sitting between two symmetrically arrayed MazF homodimers. Each MazF monomer forms a single globular domain consisting of a seven-stranded, twisted antiparallel β sheet with three α helices. The two monomers interact with each other vis hydrophobic contacts, primarily involving segments of β strand S6 and α helix H3⁸.

Results

This part was used for constructing an inducible kill-switch by AI-2 for 2020 iGEM Vilnius-Lithuania project FlavoFlow.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Christensen-Dalsgaard, M. & Gerdes, K. Translation affects YoeB and MazF messenger RNA interferase activities by different mechanisms. Nucleic Acids Research 36, 6472–6481 (2008).
2. Nigam, A., Ziv, T., Oron-Gottesman, A. & Engelberg-Kulka, H. Stress-Induced MazF-Mediated Proteins in Escherichia coli. mBio 10, e00340-19, /mbio/10/2/mBio.00340-19.atom (2019).
3. Culviner, P. H. & Laub, M. T. Global Analysis of the E. coli Toxin MazF Reveals Widespread Cleavage of mRNA and the Inhibition of rRNA Maturation and Ribosome Biogenesis. Molecular Cell 70, 868-880.e10 (2018).
4. Cho, J., Carr, A. N., Whitworth, L., Johnson, B. & Wilson, K. S. MazEF toxin-antitoxin proteins alter Escherichia coli cell morphology and infrastructure during persister formation and regrowth. Microbiology 163, 308–321 (2017).
5. Mets, T. et al. Fragmentation of Escherichia coli mRNA by MazF and MqsR. Biochimie 156, 79–91 (2019).
6. Zhang, Y. et al. MazF Cleaves Cellular mRNAs Specifically at ACA to Block Protein Synthesis in Escherichia coli. Molecular Cell 12, 913–923 (2003).
7. Vesper, O. et al. Selective Translation of Leaderless mRNAs by Specialized Ribosomes Generated by MazF in Escherichia coli. Cell 147, 147–157 (2011).
8. Kamada, K., Hanaoka, F. & Burley, S. K. Crystal Structure of the MazE/MazF Complex: Molecular Bases of Antidote-Toxin Recognition. Molecular Cell 10.