Ribosome recycling is not critical for translational coupling in Escherichia coli

We used ribosome profiling to characterize the biological role of ribosome recycling factor (RRF) in Escherichia coli. As expected, RRF depletion leads to enrichment of post-termination 70S complexes in 3′-UTRs. We also observe that elongating ribosomes are unable to complete translation because they are blocked by non-recycled ribosomes at stop codons. Previous studies have suggested a role for recycling in translational coupling within operons; if a ribosome remains bound to an mRNA after termination, it may re-initiate downstream. We found, however, that RRF depletion did not significantly affect coupling efficiency in reporter assays or in ribosome density genome-wide. These findings argue that re-initiation is not a major mechanism of translational coupling in E. coli. Finally, RRF depletion has dramatic effects on the activity of ribosome rescue factors tmRNA and ArfA. Our results provide a global view of the effects of the loss of ribosome recycling on protein synthesis in E. coli.

Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame

ABSTRACT CsrA is a global regulatory RNA binding protein that has important roles in regulating carbon metabolism, motility, biofilm formation, and numerous other cellular processes. IraD functions as an antiadapter protein that inhibits RssB-mediated degradation of RpoS, the general stress response and stationary-phase sigma factor of Escherichia coli . Here we identified a novel mechanism in which CsrA represses iraD translation via translational coupling. Expression studies with quantitative reverse transcriptase PCR, Western blotting, and lacZ fusions demonstrated that CsrA represses iraD expression. Gel mobility shift, footprint, and toeprint studies identified four CsrA binding sites in the iraD leader transcript, all of which are far upstream of the iraD ribosome binding site. Computational modeling and RNA structure mapping identified an RNA structure that sequesters the iraD Shine-Dalgarno (SD) sequence. Three open reading frames (ORFs), all of which are translated, were identified in the iraD leader region. Two of these ORFs do not affect iraD expression. However, the translation initiation region of the third ORF contains three of the CsrA binding sites, one of which overlaps its SD sequence. Furthermore, the ORF stop codon overlaps the iraD start codon, a sequence arrangement indicative of translational coupling. In vivo expression and in vitro translation studies with wild-type and mutant reporter fusions demonstrated that bound CsrA directly represses translation initiation of this ORF. We further established that CsrA-dependent repression of iraD translation occurs entirely via translational coupling with this ORF, leading to accelerated iraD mRNA decay. IMPORTANCE CsrA posttranscriptionally represses gene expression associated with stationary-phase bacterial growth, often in opposition to the transcriptional effects of the stationary-phase sigma factor RpoS. We show that CsrA employs a novel regulatory mechanism to repress translation of iraD , which encodes an antiadapter protein that protects RpoS against proteolysis. CsrA binds to four sites in the iraD leader transcript but does not directly occlude ribosome binding to the iraD SD sequence. Instead, CsrA represses translation of a short open reading frame encoded upstream of iraD , causing repression of iraD translation via translational coupling. This finding offers a novel mechanism of gene regulation by the global regulator CsrA, and since RpoS can activate csrA transcription, this also highlights a new negative-feedback loop within the complex Csr and RpoS circuitry.