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.

蛋白质合成受到纳入多肽链中的氨基酸的化学和结构特性的影响。含有连续脯氨酸的基序可减缓翻译速度并导致核糖体停滞。翻译延长因子P（EF-P）通过促进这些基序中的肽键形成，从而缓解停滞的核糖体并恢复正常的翻译速度。利用包括核糖体图谱、蛋白质组学和体内筛选在内的多种复杂技术，对各种多脯氨酸基序导致的核糖体暂停进行了深入研究，其中报告基因被整合到染色体中。然而，尚未确定引起大肠杆菌翻译暂停的基序的全谱系。在此，我们描述了一种基于质粒的双重报告系统，用于快速评估暂停基序。该报告系统包含编码mScarlet-I和氯霉素乙酰转移酶的两个耦合基因，以基于细菌荧光和存活性的方式筛选基序库。结合二丙氨酸基序库，我们使用该报告系统揭示了efp缺失的大肠杆菌菌株中不同暂停强度的基序。随后，我们使用该报告系统对四种基序库进行高通量筛选，包括在不同位置具有和没有脯氨酸的基序，通过荧光相关细胞分离（FACS）排序，并识别出影响荧光素翻译效率的新基序。本研究提供了一个体内平台，用于快速筛选影响翻译效率的氨基酸基序。