A Stress-Induced Bias in the Reading of the Genetic Code in Escherichia coli

Abstract

Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a stress-induced translation machinery (STM), composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we further characterized the STM system, finding that MazF cleaves only ACA sites located in the open reading frames of processed mRNAs, while out-of-frame ACAs are resistant. This in-frame ACA cleavage of MazF seems to depend on MazF binding to an extracellular-death-factor (EDF)-like element in ribosomal protein bS1 (bacterial S1), apparently causing MazF to be part of STM ribosomes. Furthermore, due to the in-frame MazF cleavage of ACAs under stress, a bias occurs in the reading of the genetic code causing the amino acid threonine to be encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA.

MazF activation promotes translational heterogeneity of thegrcAmRNA inEscherichia colipopulations

“…As a control, we generated the grcA ATA fluorescent reporter by substituting the ACA sequence motifs by ATA, to prevent MazF-mediated processing. In both reporters, we used an Emerald gfp version that does not contain ACA sites in the open reading frame (Em gfp ΔACA) ( Sauert, 2015 ; Oron-Gottesman et al, 2016 ), while the amino acid sequence remains the same as in the wild-type Emerald GFP ( Tsien, 1998 ).…”

“… (A) The grcA wt reporter comprises 278 nt upstream of the start codon of grcA , as well as the first 15 codons fused in frame to Em gfp ΔACA, which is a gfp variant devoid of ACA sites in the open reading frame ( Sauert, 2015 ; Oron-Gottesman et al, 2016 ). The grcA ATA reporter is a modified grcA wt reporter harboring two nucleotide substitutions (C to T) at the positions previously determined to be processed by MazF ( Vesper et al, 2011 ).…”

“…We used derivatives of E. coli strain K-12 MG1655 ( Blattner et al, 1997 ; Müller et al, 2016 ) when addressing effects of antibiotic treatments and amino acid starvation, and derivatives of E. coli strain BW27784 containing a native copy of the mazEF operon ( Khlebnikov et al, 2001 ) for experiments with arabinose-inducible systems. The open reading frame of the employed reporter gene Em gfp ΔACA is devoid of ACA sites ( Sauert, 2015 ; Oron-Gottesman et al, 2016 ), whilst the amino acid sequence corresponds to the wild-type fast-folding Emerald GFP ( Tsien, 1998 ). The chromosomally integrated mCherry reporter ( Cox III, Dunlop & Elowitz, 2010 ) under control of the phage λ promoter P R is used to infer constitutive gene expression.…”

Autoregulation of mazEF expression underlies growth heterogeneity in bacterial populations

“…the phage λ promoter drives expression of mCherry ( 33 ). A variant of the fast-folding Emerald GFP was used as an additional reporter gene, Em gfp ΔACA ( 34 ), called ‘ gfp ’ throughout the manuscript. The Em gfp ΔACA amino acid sequence corresponds to the wild-type sequence of Emerald GFP, however the nucleotide sequence was modified such that all ACA sites were substituted.…”

A Bias in the Reading of the Genetic Code of Escherichia coli is a Characteristic for Genes that Specify Stress-induced MazF-mediated Proteins

“…Previously, when the STM system was characterized [ 14 ], it was found that MazF cleaves ACA sites located in frame 0 of the coding region of the processed mRNAs, while out-of-frame ACAs are resistant to such cleavage. Moreover, under stressful conditions, when MazF cleaves in-frame ACAs, a bias in the reading of the genetic code is caused.…”