Graduate Students Mini Symposium VI 2025

13:15 h Markus Meier - AG Erb

A genetic in vitro system to dissect and engineer membrane protein insertion and translocation

Membrane proteins equip cells with diverse and crucial functions. Most of them are integrated into the membrane via the Sec translocation machinery, a highly conserved membrane complex found in all domains of life. Due to its central and essential role, Sec has been investigated for decades. However, the keystone nature of Sec has presented challenges such as toxicity in vivo and the lack of efficient analysis methods. Here, we describe a powerful in vitro system to study and engineer Sec enabling the collection of large amounts of quantitative data and eliminating the need to purify its components. By encoding the Sec machinery and implementing it into a system with cell-free protein synthesis (CFPS) functional E. coli SecYEG can be established in empty synthetic membranes. Combined with a simple luminescence assay, the system facilitates quantitative examination of translocation and insertion of model Sec substrates in real time.
The system enabled us to examine a library of 70 previously studied and 205 new variants of the central SecY protein gaining deeper insights into functionally crucial domains and revealing dozens of highly active variants with up to 8-fold increased translocation activity. Implementing the system in CFPS further improved the quality of a cell-free synthesized model membrane protein enabling not only far greater dissection of SecYEG but also setting the stage to use encoded SecYEG in in vitro synthetic biological systems.

13:45 h Joao Pedro Fernandes Queiroz - AG Shima

HcgG: an enzyme involved in [Fe]-hydrogenase cofactor biosynthesis

Hydrogenotrophic methanogenic archaea transfer electrons from H₂ to methanogenesis intermediates using a unique [Fe]-hydrogenase (Hmd) under nickel limited conditions. Unlike [NiFe] or [FeFe]-hydrogenases, Hmd lacks Fe-S cluster; instead, it contains a single iron-based cofactor called iron-guanylylpyridinol (FeGP). Hmd reduces methenyl-tetrahydromethanopterin (methenyl-H₄MPT⁺, a C₁-carrier in methanogenesis) to methylene-H₄MPT by direct transfer of a hydride derived from H₂. FeGP biosynthesis begins with the radical S-adenosylmethionine (SAM) enzyme HcgA, which forms a pyridinol precursor. This precursor is subsequently methylated by the SAM-dependent methyltransferase HcgC and conjugated to GMP by the guanylyltransferase HcgB. However, the steps from guanylylpyridinol (GP) to mature FeGP, and its incorporation into the Hmd apoenzyme, remain poorly understood. Our current model proposes that the maturases HcgE and HcgF activate the carboxyl group of GP, preparing it for three key transformations: (1) reduction of the carboxyl group to an acyl group, (2) insertion of Fe²⁺, and (3) incorporation of two CO ligands. Regardless of their order, these steps converge on the final maturation enzyme, HcgG. HcgG is a radical SAM enzyme thought to generate CO from an organic precursor and incorporate them into FeGP. In addition, HcgG may facilitate Fe²⁺ insertion—possibly with the help of HcgD, a protein with a dinuclear iron-binding site—and promote acyl group formation. Plausible intermediates of HcgG include AMP-GP, formed via the ATP-dependent reaction of HcgE, or an HcgF-GP adduct. Nevertheless, the identity of the electron donor and its interaction with partner proteins remains unknown. In this talk, I will present recent insights and ongoing efforts to elucidate the catalytic mechanism of HcgG.

14:15 h Moritz Weber - MPRG Höfer

RNAylation – a niche phenomenon or a widespread biological concept

RNAylation is a recently discovered posttranslational protein modification in which ADP‑ribosyltransferases (ARTs) covalently attach 5´-NAD-capped RNA (NAD-RNA) to proteins. To date, only one bacteriophage T4-encoded ART has been described to RNAylate its target proteins in E.coli. Since NAD-RNA and ARTs are present in all domains of life, RNAylation could be widespread in nature. This study aims to identify novel ARTs with RNAylation ability and investigate the occurrence of RNAylation in biological systems beyond the described interaction of E. coli and bacteriophage T4.
Here, we demonstrate that HopU1, an ART from Pseudomonas syringae, a phytopathogenic bacterium, can RNAylate its target protein GRP7 from Arabidopsis thaliana in vitro. We show that HopU1 modifies the same arginine residue of GRP7 in both ADP-ribosylations and RNAylations.
Our investigation of the substrate scope of HopU1 revealed that HopU1 prefers linear NAD-RNAs over NAD-RNAs with secondary structures for RNAylation. Further, we identified additional RNAylation targets of HopU1.
This work establishes HopU1 as the second described ART with RNAylation ability, suggesting that even more ARTs can catalyze RNAylations. Our results suggest the possible occurrence of RNAylation in the interaction between P. syringae and A. thaliana, representing the first described RNAylation in a eukaryotic system. These results indicate that RNAylation could be a widespread phenomenon.