Solid-state NMR for structural studies of infection-related bacterial RNAs

• Applicant: Dr. Alexander Marchanka
• Subject Area: Structural Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 510696305

In this project, we aim to develop an integrative solid-state NMR-based approach that can provide structural insights into RNAs as part of large protein-RNA complexes in bacterial infections, and we aim to apply this novel approach to understand in molecular detail how a regulatory RNA together with an abundant RNA chaperone Hfq regulates gene expression in bacteria.Large RNAs are challenging objects to study both by X-ray crystallography and electron microscopy due to their high flexibility. Solution-state NMR has an intrinsic molecular weight limit and faces severe difficulties to characterize RNA longer than 100 nucleotides. Solid-state NMR (ssNMR) is a contemporary structural biology technique that can provide atomic level of resolution for large biomolecular assemblies without intrinsic size limitations. Here we propose a ssNMR based approach for structural characterization of RNA that uses a combination of segmental and nucleotide-type specific labeling and employs a multitude of 13C-detected and 1H-detected experiments. Additionally we will apply complementary magnetic resonance techniques, including paramagnetic relaxation enhancement (PRE) ssNMR and Electron Paramagnetic Resonance (EPR) spectroscopy. Building on the methodological expertise gained in this project phase we will address the details of action mechanism of regulatory RNAs in bacterial infections. The function of many regulatory bacterial small RNAs (sRNAs) is dependent on the RNA chaperone Hfq. Hfq is highly conserved among bacteria and contains a core domain, that assembles into a stable hexameric ring with several binding sites for RNA, which all are essential for efficient sRNA binding. Moreover, Hfq binds simultaneously to sRNAs and mRNAs, stabilizing them and facilitating their base-pairing. While several X-ray structures of Hfq alone or in complex with single nucleotides or short oligonucleotides are available, only one atomic structure of Hfq in complex with an sRNA has been obtained so far and the precise molecular mechanism of Hfq-mediated sRNA-mRNA interaction remains ambiguous. Here, an ssNMR-based approach can be a perfect tool to obtain important structural and motional data on binary Hfq-sRNA and tertiary mRNA-Hfq-sRNA complexes at atomic resolution.Overall, our new approach presents a unique opportunity to investigate large bacterial RNA in close-to-native conditions. The methods developed here will enable structural studies at atomic resolution of many other infection-related RNAs.

• DFG Programme: Research Grants