Translational Control using an Expanded Genetic Code.

Yusuke Kato

doi:10.3390/ijms20040887

Abstract

A bio-orthogonal and unnatural substance, such as an unnatural amino acid (Uaa), is an ideal regulator to control target gene expression in a synthetic gene circuit. Genetic code expansion technology has achieved Uaa incorporation into ribosomal synthesized proteins in vivo at specific sites designated by UAG stop codons. This site-specific Uaa incorporation can be used as a controller of target gene expression at the translational level by conditional read-through of internal UAG stop codons. Recent advances in optimization of site-specific Uaa incorporation for translational regulation have enabled more precise control over a wide range of novel important applications, such as Uaa-auxotrophy-based biological containment, live-attenuated vaccine, and high-yield zero-leakage expression systems, in which Uaa translational control is exclusively used as an essential genetic element. This review summarizes the history and recent advance of the translational control by conditional stop codon read-through, especially focusing on the methods using the site-specific Uaa incorporation.

Highlights

Conditional induction of gene expression is one of the most important technologies for constructing synthetic gene circuits, and such regulation has applications from basic research to industrial use
We describe the control of target gene expression in vivo by conditional stop codon read-through, especially focusing on the recently advanced method using a site-specific unnatural amino acid (Uaa) incorporation system
A High-yield and Zero-Leakage Expression System (HYZEL) system using a combination of the 3-iodo-L-tyrosine incorporation system, PBAD-araO/araC regulated T7 RNA polymerase, and PT7-lacO/lacI regulated target gene, maintained an expression construct for the potent toxin colicin E3, which kills its host E. coli with a few molecules, in a multicopy plasmid carrying pBR322 replication origin [48]

Summary

Introduction

Conditional induction of gene expression is one of the most important technologies for constructing synthetic gene circuits, and such regulation has applications from basic research to industrial use. An established method for translational control is the conditional stop codon read-through (Figure 1). A mutant tRNA that recognizes the inserted stop codon (stop codon suppressor tRNA) can rescue the function of the target gene product by incorporation of an amino acid at the inserted stop codon (stop codon read-through), which results in a production of the full-length protein. The expression of a target gene can be controlled at the translational level by conditional production of an aminoacylated stop codon suppressor tRNA. We describe the control of target gene expression in vivo by conditional stop codon read-through, especially focusing on the recently advanced method using a site-specific unnatural amino acid (Uaa) incorporation system. This review does not cover a drug-stimulated translational read-through of stop codons [1]

Classifications

Thermolabile tRNACUA

Inducible Transcription of tRNACUA

Inducible Aminoacyl-tRNA Synthetase

Introduction of the tRNACUA Gene

Multiplexed TAG Insertion in a Target Gene

Location of the UAG Insertion

Uaa-Residue Conversion to Standard Amino Acid Residue

Suppression of Peptide Release Factor

Uaa-Permissive Elongation Factor

Host Selection

Uaa Transcriptional Switch