Impact of EFP on translational efficiency of specific motifs
Translation dynamics are influenced by the chemical and structural properties of the amino acids incorporated into the polypeptide chain. 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 the stalled ribosomes and enabling regular translational speed. Ribosome pausing at various polyproline motifs has been studied using labor-intensive techniques such as ribosome profiling, proteomics, and in vivo screenings with reporters incorporated into the chromosome.  Here, we describe a plasmid-based dual reporter for rapid assessment of translation efficiencies at various motifs. We demonstrate that the construction of a system with  coupled genes encoding mScarlet-I and chloramphenicol acetyltransferase enables sensitive screenings of translation efficiencies at motifs based on bacterial fluorescence and survival. Plate-based screenings of diprolyl motif libraries in an E. coli strain lacking efp (?efp) reveals sequences which have diverse translation efficiencies. Ultimately, high-throughput screening of motif libraries sorted by fluorescence-associated cell sorting (FACS) reveals that sequences lacking prolines can cause intermediate to strong ribosome pauses in ?efp. Our study offers insights into unusual ribosome stalling motifs and provides an in vivo platform for rapid screening of the translational efficiency of diverse motifs.