Mechanismus und Regulation der RNA-Entwindung durch DEAD-Box RNA-Helikasen
Zusammenfassung der Projektergebnisse
RNA helicases are an important class of enzymes that unwind RNA duplex regions in an ATP-dependent reaction, and fulfil key roles in RNA metabolism. While the molecular basis for ATP-dependent unwinding of RNA by the helicase core is well-understood, the mechanisms of regulation of helicase activity, either by domains flanking the core in cis, or by interaction partners in trans, are less clear. In the research described here, we have addressed this question using three different model systems: a monomeric DEAD-box protein containing a helicase core and an RNA-binding domain (RBD; Aim 1), a dimeric DEAD-box helicase containing two helicase cores and two RBDs (Aim 2), and a helicase consisting of an isolated core that is regulated by a set of interaction partners (Aim 3). We show that RNA binding to the RBD can activate the helicase core through an allosteric mechanism in the monomeric DEAD-box protein YxiN, but not in the dimeric counterpart Hera from T. thermophilus, illustrating that regulatory mechanisms may not be universal. We also show that the two RBDs in Hera can functionally cooperate with both helicase cores, although the cooperation with the helicase core on the same protomer is more efficient. Further mechanistic studies on the cooperation of the two RBDs and the two cores in Hera require larger, physiologically relevant RNA substrates. In CLIP- experiments in Thermus, we have identified tRNAs as promising RNA substrates for such studies. These experiments also showed that Hera acts as a general chaperone in T. thermophilus, and interacts with RNAs with a high propensity to form secondary structures after cold-shock. We analyzed intermolecular activation of helicases by dissecting the contributions of individual domains of the translation initiation factors eIF4B and 4G, and the role of the interaction of the 5’-cap and 4E, for the activation of eIF4A. Finally, we addressed the role of the mRNA itself in eIF4A activation, and paved the way for systematic studies of eIF4A conformational dynamics and translation efficiencies under identical conditions. The results we obtained have provided important insight into the regulation of DEAD-box helicase activity. They have led to a number of novel hypotheses that we and others will study in the future, and have undoubtedly provided a valuable contribution to the field.
Projektbezogene Publikationen (Auswahl)
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(2019) Single-stranded regions modulate conformational dynamics and ATPase activity of eIF4A to optimize 5’-UTR unwinding, Nucleic Acids Res. 47(10):5260-5275
Andreou, A.Z. Harms, U. & Klostermeier, D.
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(2020) The Thermus thermophilus DEAD-box protein Hera is a general RNA binding protein and plays a key role in tRNA metabolism, RNA 26(11):1557-1574
Donsbach, P., Yee, B.A., Sanchez-Hevia, D., Berenguer, J., Aigner, S., Yeo, G.W., Klostermeier, D.
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(2021) “Probing RNA helicase Conformational Changes by Single-Molecule FRET Microscopy Probing RNA helicase mechanisms using single-molecule fluorescence methods, Special Issue “Single molecule approaches: watching DNA repair one molecule at a time”, Methods Mol. Biol.2209:119-132
Krause, L., Klostermeier, D.
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(2021), “Regulation of RNA helicase activities: Principles and case studies, Special Issue “Structure, mechanism, function and regulation of RNA helicases”, Biol. Chem. 402(5):529-559
Donsbach, P., Klostermeier, D.
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(2022) „Determining rate constants for conformational changes in RNA helicases by single-molecule FRET TIRF microscopy” for the Methods Special Issue “Experimental Techniques and Strategies for the Analysis of DNA Helicases and RNA Helicases”, S1046- 2023(22)00071-8
Chakraborty, A., Krause, L., Klostermeier D.
