A bacterial regulatory uORF senses multiple classes of ribosome-targeting antibiotics

Abstract

Long 5′ UTRs in bacteria often contain regulatory elements that modulate expression of the downstream gene in response to environmental stimuli. In most examples of such regulation, the mechanism involves switching between alternative 5′ UTR RNA structures that impact transcription, stability, or translation of the mRNA. Here, we show that transcription of the Escherichia coli topAI gene is prematurely terminated by the termination factor Rho under standard laboratory growth conditions, and that this occurs as a result of translational repression. Regulation of topAI translation is controlled by a sensory ORF, toiL, located within the topAI 5′ UTR. We show that ribosomes translating toiL stall in a sequence-specific manner in the presence of specific ribosome-targeting antibiotics. Ribosome stalling at toiL induces conformational changes in the RNA structure of the topAI 5′ UTR, unmasking the topAI ribosome-binding site, thereby relieving translational repression and preventing premature transcription termination. Thus, toiL acts as a sensor of translation stress, leading to regulation of topAI at both the translational and transcriptional levels.

Regulatory roles of Escherichia coli 5' UTR and ORF-internal RNAs detected by 3' end mapping

“…Among the 3´ ends in 5´ UTRs, several correspond to sites of known cis -acting RNA regulation (annotated in Supplementary file 3 ). These include mRNAs that previously have been shown to be regulated by premature Rho-dependent termination, such as the riboswitch-regulated genes thiM ( Bastet et al, 2017 ; Figure 2—figure supplement 1A ), mgtA ( Hollands et al, 2012 ), ribB ( Hollands et al, 2012 ) and lysC ( Bastet et al, 2017 ), and the translationally-repressed genes ilvX ( Lawther and Hatfield, 1980 ) and topAI ( Baniulyte and Wade, 2019 ). We also noticed that even some 5´ UTRs where regulation has only been reported to be at the level of translation, such as the RNA thermometer upstream of rpoH ( Morita et al, 1999a ; Morita et al, 1999b ) and at ribosomal protein operons (reviewed in Zengel and Lindahl, 1994 ), harbored defined 3´ ends in 5´ UTRs.…”

Regulatory roles of 5′ UTR and ORF-internal RNAs detected by 3′ end mapping

“…Among the 3′ ends in 5′ UTRs, several correspond to sites of known cis -acting RNA regulation (annotated in Supplementary file 3). These include mRNAs that previously have been shown to be regulated by premature Rho-dependent termination, such as the riboswitch-regulated genes thiM (Bastet et al, 2017) (Figure 2—figure supplement 1A), mgtA (Hollands et al, 2012), ribB (Hollands et al, 2012) and lysC (Bastet et al, 2017), and the translationally-repressed genes ilvX (Lawther and Hatfield, 1980) and topAI (Baniulyte and Wade, 2019). We also noticed that even some 5′ UTRs where regulation has only been reported to be at the level of translation, such as the RNA thermometer upstream rpoH (Morita et al, 1999a; Morita et al, 1999b) and at ribosomal protein operons (reviewed in (Zengel and Lindahl, 1994)), harbored defined 3′ ends in 5′ UTRs.…”

Regulatory Cross Talk between Motility and Interbacterial Communication in Salmonella enterica Serovar Typhimurium

“…All luciferase (luxCDABE from Photorhabdus luminescens) translational reporter fusions were constructed in plasmid pGB135 (Baniulyte and Wade, 2019). Wild-type and mutant upstream regions, including the putative promoter and extending to the annotated start codon, were PCR-amplified from S. Typhimurium LT2 genomic DNA using oligonucleotides listed in Table S4.…”