Genome-wide effects of the antimicrobial peptide apidaecin on translation termination in bacteria

Abstract

Biochemical studies suggested that the antimicrobial peptide apidaecin (Api) inhibits protein synthesis by binding in the nascent peptide exit tunnel and trapping the release factor associated with a terminating ribosome. The mode of Api action in bacterial cells had remained unknown. Here genome-wide analysis reveals that in bacteria, Api arrests translating ribosomes at stop codons and causes pronounced queuing of the trailing ribosomes. By sequestering the available release factors, Api promotes pervasive stop codon bypass, leading to the expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to the futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate the development of new medicines and research tools for genome exploration.

First-principles model of optimal translation factors stoichiometry

“…This upper bound is still much smaller than the reported median initiation time, suggesting that the queuing factor for termination is small. As additional support to the view that translation is far from being termination limited, small that queues at stop codons are S4 only globally observed in ribosome profiling upon severe perturbations [3,33,42,58] With regards to translation elongation, transient queuing in the body of gene can also lead to a difference between molecular and coarse-grained transition times in our model. However, the fraction of ribosomes transiently stalled due to this queuing scales as τ in the low density phase (defined by requirements τ < 1 and τ < (1 + √︀ ℓ ) −1 ≈ 0.25) of the TASEP model [64].…”

“…The resulting nonlinearity would forbid the decoupling in the optimization procedure between RFI and RF4. Although absolute rates of termination are difficult to measure in vivo, translation on mRNAs is generally thought to be limited at the initiation step [36], and consistently, ribosome queuing at stop codons in bacteria is not usually observed (except under severe perturbations, e.g., [3,27,33,42,58]). In the physiological regime of fast termination, the queuing factor converges to 1, yielding simple solutions that depend only on biophysical parameters (equations 7).…”

Identification of novel translated small ORFs in Escherichia coli using complementary ribosome profiling approaches

“…active stop codons. However, apidaecin treatment does not cause ribosomes to be exclusively associated with stop codons; ribosomes are also enriched in the regions immediately upstream and downstream of stop codons, and at start codons (19), such that stop codon identification from Ribo-Api/Pmn data is associated with a high FDR (estimated at 33.7% for our analysis). Moreover, identification of a stop codon position rarely indicates the position of the associated start codon(s).…”

“…TERFs were identified as described for IEFRs, but using Ribo-Api/Pmn data (NCBI SRA accession number SRR11728142) (19).…”

“…Ribosome profiling has also been applied to E. coli cells treated with the antibiotic apidaecin, which traps ribosomes at stop codons by binding in the nascent peptide exit tunnel and trapping release factor (18). This method, "Ribo-Api", also leads to ribosome accumulation upstream and downstream of stop codons, and at start codons (19); this additional signal can be partially reduced by addition of puromycin to cell lysates ("Ribo-Api/Pmn"), but even with this reduction in signal away from stop codons, Ribo-Api/Pmn data cannot be used to identify stop codons with high specificity. Nonetheless, we reasoned that combining Ribo-RET and Ribo-Api/Pmn datasets for E. coli would facilitate the identification of translated sORFs with high sensitivity, since the likelihood of detecting Ribo-RET signal at a start codon and Ribo-Api/Pmn signal at the corresponding stop codon by chance would be very low.…”