Enhanced translation of leaderless mRNAs under oxidative stress in Escherichia coli

Abstract

The bacterial response to oxidative stress requires the adaptation of the proteome to the hostile environment. It has been reported that oxidative stress induces a strong and global inhibition of both, transcription and translation. Nevertheless, whereas it is well known that transcription of a small group of genes is induced thanks to transcription factors such as OxyR and SoxR, an equivalent mechanism has not been described for translation. Here we report that whereas canonical translation that depends on Shine Dalgarno recognition is inhibited by oxidative stress in Escherichia coli, the translation of leaderless mRNA (lmRNA) is enhanced under such conditions. Both, inhibition of canonical translation and enhancement of lmRNA translation, depend on the production of (p)ppGpp. We propose that such a mechanism would allow bacteria to rapidly adapt their proteome to hostile conditions and is, perhaps, a general strategy to confront strong stressful conditions.

Oxidative stress strongly restricts the effect of codon choice on the efficiency of protein synthesis in Escherichia coli

“…In fact, our previous work has shown that addition of four codons is enough to report differences in the efficiency of translation of those codons ( Elgamal et al, 2014 ; Rojas et al, 2018 ; Leiva et al, 2020 ). Also, we have observed that under these conditions the stability of different segments of the reporter mRNA is similar ( Leiva et al, 2021 ). Therefore, we expect that changes in transcript level or alterations of translation initiation will affect the synthesis of GFP and mCherry in a similar manner.…”

“…As mCherry translation initiated by the same mechanism in all reporters, we use it as an internal control for normalization. Although lmRNA translation of most genes is less efficient than SD translation under non-stress conditions, we have recently observed that under oxidative stress induced by 250 μM paraquat, lmRNA translation is activated ( Leiva et al, 2021 ). Using these new set of SD reporters, we observed that changes in the selection of Leu codons added to the vector led to a 6.4-fold difference in GFP synthesis under non-stress conditions ( Figure 3 ).…”

“…Some of these reporters have been used in previous studies ( Rojas et al, 2018 ; Leiva et al, 2020 ). Furthermore, annealed oligonucleotides enriched for the CTC and CTG codons (oligonucleotides 59 and 61 in Supplementary Table S1 ) were inserted into pSD and plmRNA plasmids ( Leiva et al, 2021 ), two variants of pBAD30SFIT designed to generate a leader transcript (42-nucleotides containing an SD sequence) and a leaderless transcript, respectively. Secondary structure of segments of the 5′ end of the produced mRNAs (57, 100, and 140 nucleotides long) were predicted using NUPACK with the default parameters ( Zadeh et al, 2011 ).…”

“…Global alterations to translation have also been observed. For example, we found that the increase in (p)ppGpp levels under oxidative stress suppresses Shine-Dalgarno (SD) led translation, while enhancing translation of genes with the initiation codon at or very near to the 5′ end of mRNA (leaderless mRNAs or lmRNAs; Leiva et al, 2021 ). In addition to regulation of translation initiation, elongation is also known to be altered under oxidative stress.…”

“…Some of the aforementioned studies induce oxidative stress using 250 μM paraquat ( Blanchard et al, 2007 ; Leiva et al, 2020 , 2021 ), a cycling agent that oxidizes intracellular redox cofactors such as NADPH and partially reduces oxygen to superoxide radical ( Hassan and Fridovich, 1979 ). Together, these studies have shown that when exposed to 250 μM paraquat, E. coli MG1655 changes its transcriptional program ( Blanchard et al, 2007 ), inhibits SD-led translation while enhancing translation of lmRNA ( Leiva et al, 2021 ) and alters translation of Gly codons ( Leiva et al, 2020 ). Nevertheless, how the translation of the remaining codons is affected is currently unknown.…”