Versatile Dual\nReporter to Identify Ribosome Pausing\nMotifs Alleviated by Translation Elongation Factor P

Protein synthesis is influenced by the chemical and structural\nproperties of the amino acids incorporated into the polypeptide chain.\nMotifs containing consecutive prolines can slow the translation speed\nand cause ribosome stalling. Translation elongation factor P (EF-P)\nfacilitates peptide bond formation in these motifs, thereby alleviating\nstalled ribosomes and restoring the regular translational speed. Ribosome\npausing at various polyproline motifs has been intensively studied\nusing a range of sophisticated techniques, including ribosome profiling,\nproteomics, and in vivo screening, with reporters incorporated into\nthe chromosome. However, the full spectrum of motifs that cause translational\npausing in Escherichia coli has not\nyet been identified. Here, we describe a plasmid-based dual reporter\nfor rapid assessment of pausing motifs. This reporter contains two\ncoupled genes encoding mScarlet-I and chloramphenicol acetyltransferase\nto screen motif libraries based on both bacterial fluorescence and\nsurvival. In combination with a diprolyl motif library, we used this\nreporter to reveal motifs of different pausing strengths in an E. coli strain lacking <i>efp</i>. Subsequently,\nwe used the reporter for a high-throughput screen of four motif libraries,\nwith and without prolines at different positions, sorted by fluorescence-associated\ncell sorting (FACS) and identify new motifs that influence the translational\nefficiency of the fluorophore. Our study provides an in vivo platform\nfor rapid screening of amino acid motifs that affect translational\nefficiencies.