Browsing by Author "Artsimovitch, Irina"

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    Allosteric couplings upon binding of RfaH to transcription elongation complexes
    (2022) Alejandro Molina, Jose; Galaz-Davison, Pablo; Komives, Elizabeth A.; Artsimovitch, Irina; Ramirez-Sarmiento, Cesar A.
    In every domain of life, NusG-like proteins bind to the elongating RNA polymerase (RNAP) to support processive RNA synthesis and to couple transcription to ongoing cellular processes. Structures of factor-bound transcription elongation complexes (TECs) reveal similar contacts to RNAP, consistent with a shared mechanism of action. However, NusG homologs differ in their regulatory roles, modes of recruitment, and effects on RNA synthesis. Some of these differences could be due to conformational changes in RNAP and NusG-like proteins, which cannot be captured in static structures. Here, we employed hydrogen-deuterium exchange mass spectrometry to investigate changes in local and non-local structural dynamics of Escherichia coli NusG and its paralog RfaH, which have opposite effects on expression of xenogenes, upon binding to TEC. We found that NusG and RfaH regions that bind RNAP became solvent-protected in factor-bound TECs, whereas RNAP regions that interact with both factors showed opposite deuterium uptake changes when bound to NusG or RfaH. Additional changes far from the factor-binding site were observed only with RfaH. Our results provide insights into differences in structural dynamics exerted by NusG and RfaH during binding to TEC, which may explain their different functional outcomes and allosteric regulation of transcriptional pausing by RfaH.
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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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    Metamorphic proteins and how to find them
    (2024) Porter, Lauren L.; Artsimovitch, Irina; Ramirez-Sarmiento, Cesar A.
    In the last two decades, our existing notion that most foldable proteins have a unique native state has been challenged by the discovery of metamorphic proteins, which reversibly interconvert between multiple, sometimes highly dissimilar, native states. As the number of known metamorphic proteins increases, several computational and experimental strategies have emerged for gaining insights about their refolding processes and identifying unknown metamorphic proteins amongst the known proteome. In this review, we describe the current advances in biophysically and functionally ascertaining the structural interconversions of metamorphic proteins and how coevolution can be harnessed to identify novel metamorphic proteins from sequence information. We also discuss the challenges and ongoing efforts in using artificial intelligence-based protein structure prediction methods to discover metamorphic proteins and predict their corresponding three-dimensional structures.