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 containing consecutive prolines can slow the 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 the 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 screening, with reporters incorporated into
the chromosome. However, the full spectrum of motifs that 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 used this
reporter to reveal motifs of different pausing strengths in an E. coli strain lacking efp. Subsequently,
we used 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 the translational
efficiency of the fluorophore. Our study provides an in vivo platform
for rapid screening of amino acid motifs that affect translational
efficiencies.