Biology subjects

Krafczyk, R.

Publications and source records attributed to Krafczyk, R..

3 recordsLinked to original sources

bioRxiv2024

EF-P and its paralog EfpL (YeiP) differentially control translation of proline containing sequences

Polyproline sequences (XPPX) stall ribosomes, thus being deleterious for all living organisms. In bacteria, translation elongation factor P (EF-P) plays a crucial role in overcoming such arrests. 12% of eubacteria possess an EF-P paralog - YeiP (EfpL) of unknown function. Here, we functionally and structurally characterize EfpL from Escherichia coli and demonstrate its yet unrecognized role in the translational stress response. Through ribosome profiling, we analyzed the EfpL arrest motif spectrum and discovered additional stalls beyond the canonical XPPX motifs at single-proline sequences (XPX), that both EF-P and EfpL can resolve. Notably, the two factors can also induce pauses. We further report that, contrary to the housekeeping EF-P, EfpL can sense the metabolic state of the cell, via lysine acylation. Together, our work uncovers a new player in ribosome rescue at proline-containing sequences, and provides evidence that co-occurrence of EF-P and EfpL is an evolutionary driver for higher bacterial growth rates.

microbiology↗

bioRxiv2023

Decrypting the functional design of unmodified translation elongation factor P

Stalling of ribosomes during polypeptide synthesis due to consecutive proline motifs is a challenge faced by organisms across all kingdoms. To overcome this, bacteria employ translation elongation factor P (EF-P), while archaea and eukaryotes rely on a/eIF5A. Typically, these elongation factors become active only after undergoing post-translational modifications (PTMs) such as {beta}-lysinylation, (deoxy-)hypusinylation, rhamnosylation, or 5-aminopentanolyation. An exception to this rule is found in EF-P members of the PGKGP-subfamily, which remain unmodified. However, the mechanism behind the ability of certain bacteria to avoid metabolically and energetically costly PTMs, while retaining active EF-P, remains unclear. In this study, we investigated the design principles governing the full functionality of unmodified EF-Ps in Escherichia coli. We first screened for naturally unmodified EF-Ps that are active in an E. coli reporter strain. We identified EF-P from Rhodomicrobium vannielii capable of rescuing the growth deficiencies and changes in the proteome of an E. coli {Delta}epmA mutant lacking the gene for the modifying EF-P-(R)-{beta}-lysine ligase. We then identified specific amino acids in domain I of the unmodified EF-P variant that affected its activity. Ultimately, we transferred these functional properties to other marginally active members of the PGKGP EF-P subfamily, resulting in fully functional unmodified variants in E. coli. These results have implications for the improved heterologous expression of polyproline-containing proteins in E. coli and offer applications in other bacterial hosts. Understanding the mechanisms that underlie the functionality of unmodified EF-P provides insights into cellular adaptations to optimize protein synthesis.

microbiology↗

bioRxiv2017

Structural basis for EarP-mediated arginine glycosylation of translation elongation factor EF-P

Glycosylation is a universal strategy to post-translationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann-folds, thus constituting a GT-B glycosyltransferase. While TDP-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the EF-P via its KOW-like N-domain. Based on our structural data combined with an in vitro /in vivo enzyme characterization, we propose a mechanism of inverting arginine glycosylation. As EarP is essential for pathogenicity in P. aeruginosa our study provides the basis for targeted inhibitor design.

biochemistry↗