Tomasiunaite, U. | bioRxiv Science

bioRxiv2024

A versatile dual reporter to identify ribosome pausing motifs alleviated by translation elongation factor P

Protein synthesis is influenced by the chemical and structural properties of the amino acids incorporated into the polypeptide chain. Motifs with consecutive prolines can slow down translation speed and cause ribosome stalling. Translation elongation factor P (EF-P) facilitates peptide bond formation in these motifs, thereby alleviating stalled ribosomes and restoring regular translational speed. Ribosome pausing at various polyproline motifs has been intensively studied using a range of sophisticated techniques, including ribosome profiling, proteomics, and in vivo screenings with reporters incorporated into the chromosome. However, the full spectrum of motifs which cause translational pausing in Escherichia coli has not yet been identified. Here we describe a plasmid-based dual reporter for rapid assessment of pausing motifs. This reporter contains two coupled genes encoding mScarlet-I and chloramphenicol acetyltransferase to screen motif libraries based on both bacterial fluorescence and survival. In combination with a diprolyl motif library, we use this reporter to reveal motifs of different pausing strengths in an E. coli strain lacking efp. Subsequently, we use the reporter for a high-throughput screen of four motif libraries, with and without prolines at different positions, sorted by fluorescence-associated cell sorting (FACS) and identify new motifs that influence translational efficiency of the fluorophore. Our study provides an in vivo platform for rapid screening of amino acid motifs that affect translational efficiencies.

molecular biology↗

bioRxiv2023

Decrypting the functional design of unmodified translation elongation factor P

Stalling of ribosomes during polypeptide synthesis due to consecutive proline motifs is a challenge faced by organisms across all kingdoms. To overcome this, bacteria employ translation elongation factor P (EF-P), while archaea and eukaryotes rely on a/eIF5A. Typically, these elongation factors become active only after undergoing post-translational modifications (PTMs) such as {beta}-lysinylation, (deoxy-)hypusinylation, rhamnosylation, or 5-aminopentanolyation. An exception to this rule is found in EF-P members of the PGKGP-subfamily, which remain unmodified. However, the mechanism behind the ability of certain bacteria to avoid metabolically and energetically costly PTMs, while retaining active EF-P, remains unclear. In this study, we investigated the design principles governing the full functionality of unmodified EF-Ps in Escherichia coli. We first screened for naturally unmodified EF-Ps that are active in an E. coli reporter strain. We identified EF-P from Rhodomicrobium vannielii capable of rescuing the growth deficiencies and changes in the proteome of an E. coli {Delta}epmA mutant lacking the gene for the modifying EF-P-(R)-{beta}-lysine ligase. We then identified specific amino acids in domain I of the unmodified EF-P variant that affected its activity. Ultimately, we transferred these functional properties to other marginally active members of the PGKGP EF-P subfamily, resulting in fully functional unmodified variants in E. coli. These results have implications for the improved heterologous expression of polyproline-containing proteins in E. coli and offer applications in other bacterial hosts. Understanding the mechanisms that underlie the functionality of unmodified EF-P provides insights into cellular adaptations to optimize protein synthesis.

microbiology↗