Browsing by Author "Hilal, Tarek"

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    Concerted transformation of a hyper-paused transcription complex and its reinforcing protein
    (2024) Zuber, Philipp K.; Said, Nelly; Hilal, Tarek; Wang, Bing; Loll, Bernhard; Gonzalez-Higueras, Jorge; Ramirez-Sarmiento, Cesar A.; Belogurov, Georgiy A.; Artsimovitch, Irina; Wahl, Markus C.; Knauer, Stefan H.
    RfaH, a paralog of the universally conserved NusG, binds to RNA polymerases (RNAP) and ribosomes to activate expression of virulence genes. In free, autoinhibited RfaH, an alpha-helical KOW domain sequesters the RNAP-binding site. Upon recruitment to RNAP paused at an ops site, KOW is released and refolds into a beta-barrel, which binds the ribosome. Here, we report structures of ops-paused transcription elongation complexes alone and bound to the autoinhibited and activated RfaH, which reveal swiveled, pre-translocated pause states stabilized by an ops hairpin in the non-template DNA. Autoinhibited RfaH binds and twists the ops hairpin, expanding the RNA:DNA hybrid to 11 base pairs and triggering the KOW release. Once activated, RfaH hyper-stabilizes the pause, which thus requires anti-backtracking factors for escape. Our results suggest that the entire RfaH cycle is solely determined by the ops and RfaH sequences and provide insights into mechanisms of recruitment and metamorphosis of NusG homologs across all life.
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    CryoEM analysis of small plant biocatalysts at sub-2 Å resolution
    (2022) Dimos, Nicole; Helmer, Carl P. O.; Chanique, Andrea M.; Wahl, Markus C.; Kourist, Robert; Hilal, Tarek; Loll, Bernhard
    Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial in order to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryoEM) has revolutionized structural biology, its applicability to high-resolution structural analysis of comparatively small enzymes has so far been largely unexplored. Here, it is shown that cryoEM can reveal the structures of plant borneol dehydrogenases of similar to 120 kDa at or below 2 angstrom resolution, paving the way for the rapid development of new biocatalysts that can provide access to bioactive terpenes and terpenoids.