Title: Structural dynamics of the yeast spliceosome during its activation and catalysis of splicing
The spliceosome catalyzes pre-mRNA splicing and undergoes a cascade of major structural rearrangements during the evolution of its active sites. The aims of our project are to (i) reconstitute from purified components the activation step of the yeast spliceosome – i.e., the transition from the pre-catalytic B to the activated Bact complex – and investigate the regulation of Brr2 RNA helicase in this step, (ii) chart the spliceosome’s dynamic RNA/RNP interaction networks during its activation step and its catalytic phase via biochemical/biophysical methods and (iii) elucidate the spliceosome’s 3D structure at defined stages of its function using electron cryomicroscopy.
Title: Crystallographic studies on molecular motors of the spliceosome
Eight different DExD/H-box ATPases promote the structural and compositional changes required for the assembly, activation and disassembly of the spliceosome. The activities of the DEAH-box proteins Prp2 and Prp43 in the spliceosome are specifically triggered by direct interaction with the intrinsically disordered G-patch domains of the proteins Spp2 and Ntr1, respectively. We aim to unravel the structural basis for the strong stimulation of ATPase and helicase activities of Prp43 upon binding of the Ntr1 G-patch domain, as well as the impact of Spp2 on the Prp2-dependent formation of the catalytically active spliceosome.
Title: Timing and trajectory of tRNA movement through the ribosome
The dynamics of elongation factor G (EF-G)-promoted tRNA–mRNA translocation through the ribosome will be studied using a toolbox of fluorescence labels developed during the first funding period. We will study the the processivity of the ribosome and its mRNA unwinding activity, translocation during recoding events such as programmed -1 frameshifting and bypassing, and the interplay between EF-G and FusB, a factor conferring resistance against the antibiotic fusidic acid in Staphylococcus aureus.
Title: 3D structure determination of dynamic macromolecular complexes by single particle electron cryomicroscopy
Single particle cryo-EM is a powerful method to study the 3D structure of large and dynamic macromolecules that cannot be purified in amounts needed for crystallization. We have recently developed a suite of methods to achieve improved biochemical quality of macromolecular complexes of low abundance. With these tools in hand we are now in a position to obtain reliable high-resolution structure information of macromolecular complexes that are known to be rather fragile and dynamic. We want to determine 3D structures of these dynamic macromolecular complexes at the highest possible resolution by taking conformational flexibility and variations in structural composition into account. Specifically, our work will be focused on the yeast spliceosome, the 70S ribosome, the pyruvate dehydrogenase complex and the nuclear export protein CRM1.
Title: Single-molecule fluorescence spectroscopy of the structure and dynamics of macromolecular assemblies
We will apply two new techniques, Metal Induced Energy Transfer (MIET) and cryo Single Molecule Localization Microscopy (cryoSMLM), to elucidate structural details of macromolecular complexes with nanometer resolution. MIET uses the extreme sensitivity of a dye’s fluorescence lifetime on its distance from a metal surface, which allows for determining its distance from that surface with nanometer accuracy. CryoSMLM at temperatures around 90 K allows to measure lateral distances between fluorescently labelled sites with sub-nanometer resolution. Both methods have the potential to revolutionize the applicability of single-molecule fluorescence detection for structural biology
Title: Interactions oft eh essential RNase Y in Bacillus subtilis
In bacteria, the RNA degradosome is the major machinery for the determination of mRNA half-lives. Therefore, it is essential to understand how this machine operates. We have discovered RNase Y, the central component of the RNA degradosome in Bacillus subtilis. In the second funding period, we want to study both the structure and the function of the degradosome with a special focus on RNase Y and its interactions. A major effort will be devoted to the crystallization of RNase Y and/or its functional domains as well as of complexes between the degradosome proteins.
Title: Structural and functional dynamics of slam RNA-protein particles in Drosophila embryos
Several mRNAs contain a non-coding function, e. g. specific subcellular localization, besides their function in encoding proteins. Slam and oskar mRNAs are unique among mRNAs with a non-coding function, in that the encoded protein is required for proper subcellular localization of the mRNA and that mRNA and protein form a complex. In the proposed project, the structural basis of the RNA-protein complex will be investigated. Furthermore the functional relevance of the mutual dependence of mRNA and its protein for Slam's role in formation and polarization of the plasma membrane will be addressed.
Title: Mass-spectrometry-based molecular probing studies on protein (-ligand) complexes
We will investigate protein–protein and protein–ligand complexes by mass-spectrometric methods that involve: (i) the use of photo-reactive protein crosslinking reagents that do not suffer from being restricted to the reactive lysine residues used frequently in the past; (ii) quantitative protein–protein crosslinking, which will allow us to observe changes in the tertiary and quaternary structure of protein complexes in their various assembly states, (iii) the mass-spectrometric investigation of protein–RNA contact sites after crosslinking, and (iv) the development of an MS-based method for sequencing larger crosslinked RNA moieties.
Title: The role of the DEAD-box helicase Dbp5p and the Fe/S-protein Rli1p in translation termination
Eukaryotic translation termination involves the eukaryotic release factors eRF1 (Sup45) and eRF3 (Sup35). In S. cerevisiae novel termination factors were identified: The DEAD-box RNA helicase Dbp5/Rat8, its cofactor Gle1, and the iron-sulfur-containing ABC-family ATPase Rli1. Our goal is to understand the functions of these additional translation termination factors and uncover how they coordinate their actions to terminate translation. For this purpose we use biochemical interaction studies and genetic experiments in vivo and generate purified components to establish an in vitro translation system from S. cerevisiae.
Title: RNA elements required for pre-ribosome remodelling and pre-rRNA processing in 60S biogenesis in yeast
Ribosome biogenesis in eukaryotes is a major cellular pathway that requires multiple cofactors, among them DEAD-box RNA helicases. We have identified binding sites of multiple large subunit RNA helicases on yeast ribosomal RNA and will analyse their molecular functions in the pathway. Interestingly, we also find novel interactions with various cellular RNAs for several RNA helicases, indicating that they perform additional functions in other pathways. Understanding the molecular functions and regulation of RNA helicases will shed light on key steps in RNA metabolism and on the interplay of different pathways.
Title: lntegrated structural biology of the Mediator complex
Integrated structural biology of the Mediator complex Mediator is a 25-subunit, 1.4 Megadalton complex that acts as the central coactivator for RNA polymerase (Pol) II transcription of protein-coding genes in eukaryotic cells. The aim of this project is to solve the hybrid structure of the core Mediator complex with an integrated structural biology approach that requires, in addition to X-ray crystallography, cryo-electron microscopy (cryo-EM), crosslinking coupled to mass spectrometry (XL-MS), and molecular modeling in collaborations with other SFB teams. Expected results will be of fundamental importance for understanding transcription initation and will elucidate the enigmatic mechanisms of gene regulation at promoters.
Prof. Dr. Ralf Ficner
Telfon: +49 (0)551-39-14072
Fax: +49 (0)551-39-14082
Susanne van Beckum
Tel.: +49 (0)551-39-10972
Fax: +49 (0)551-39-14082
SFB860 Georg-August-Universität Göttingen
Abtl. Molekulare Strukturbiologie