Molecular evolution of elongation factor P in Escherichia coli
Final Report Abstract
This research project aimed to gain a molecular understanding of the natural variations of the elongation factor P (EF-P) and to generate an evolved variant for the heterologous expression of polyproline-containing proteins in Escherichia coli. During the translation of two or more consecutive prolines, a ribosome pause occurs that can be alleviated by EF-P. EF-P is normally only active after post-translational modification. For example, EF-P is ß-lysylated in E. coli, which is widely used as a chassis for biotechnological applications, for which the activity of two enzymes, L-lysine-2,3-aminomutase EpmB and (R)-ß-lysine ligase EpmA, is required. While only a moderate number of polyproline motifs occur in E. coli, the number of these motifs exceeds the total number of proteins encoded in Actinobacteria. In particular, the polyproline motifs are accumulated in proteins of the secondary metabolism, e.g. for the synthesis of polyketides in Streptomyces species. This project was based on our previous results, according to which Streptomyces and other Actinobacteria use an EF-P that is functional without any post-translational modification, and thus compensates for this burden. Characteristic of this type of EF-P is a rigid loop (Pro-Gly-Lys-Gly-Pro), in which the normally modified amino acid is embedded. This loop gave the EF-P PGKGP subfamily its name. In the course of the project, we succeeded in: 1. finding a naturally occurring unmodified EF-P that is fully functional in E. coli, 2. using targeted mutagenesis to find out why this EF-P of the PGKGP subfamily is active and 3. converting a naturally occurring unmodified EF-P that is inactive in E. coli into an active variant by applying the design rules developed in (2). In a second part, we developed an in vivo reporter system that visualizes the strength of ribosome pauses in translation in a high-throughput manner and is suitable for any bacterial translation factor. With these results, we have not only expanded the toolbox for synthetic biology, but also gained new functional and mechanistic insights into the fundamental process of translation.
Publications
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Decrypting the functional design of unmodified translation elongation factor P. Cell Reports, 43(5), 114063.
Tomasiunaite, Urte; Kielkowski, Pavel; Krafczyk, Ralph; Forné, Ignasi; Imhof, Axel & Jung, Kirsten
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Versatile Dual Reporter to Identify Ribosome Pausing Motifs Alleviated by Translation Elongation Factor P. ACS Synthetic Biology, 13(11), 3698-3710.
Tomasiunaite, Urte; Brewer, Tess; Burdack, Korinna; Brameyer, Sophie & Jung, Kirsten
