Fig. 3: The function of EfpL in alleviation of ribosome stalling.
From: EF-P and its paralog EfpL (YeiP) differentially control translation of proline-containing sequences
A Scheme of the in vivo stalling reporter system33. The system operates on the histidine biosynthesis operon of E. coli. In its natural form, the histidine biosynthesis gene cluster is controlled by the His-leader peptide (HisL), which comprises seven consecutive histidines. In our setup, the original histidine residues (His1 through His4) were replaced by artificial sequence motifs (XXX). Non-stalling sequences promote the formation of an attenuator stem loop (upper part) that impedes transcription of the downstream genes, thus ultimately preventing light emission. Conversely, in the presence of an arrest motif, ribosomes pause and hence an alternative stem loop is formed that does not attenuate transcription of the luxCDABE genes of Photorhabdus luminescens. B In vivo comparison of pausing at PPN in E. coli (for strain labeling and immunoblotting details see (A)). Pausing strength is given in relative light units (RLU) (n = 12, biological replicates, mean with sd indicated as error bars). Statistically significant differences according to an ordinary one-way ANOVA (*P < 0.0332, ****P < 0.0001, ns not significant). C Scheme of the in vitro cell-free stalling reporter assay. The system is based on nanoluc luciferase (nluc®) which is preceded by an artificial sequence motif (XXX). DNA is transcribed from a T7 promoter (PT7) using purified T7 polymerase (NEB). Pausing strength is proportional to light emission. D In vitro transcription and translation of the nLuc® variant nLuc_PPN. The absence (no factor) or presence of the respective translation elongation factors of E. coli (EF-P, EfpL, EfpL_R33K) is shown. Translational output was determined by measuring bioluminescence in a time course of 15 min and endpoints are given in relative light units (RLU/min±sd) (n ≥ 3, technical replicates). Statistically significant differences to the control (no factor) according to ordinary one-way ANOVA (**P = 0.0015, ***P = 0.0005, ns not significant). B, D Source data are provided as a Source Data file.
