Oral Presentation 50th Lorne Proteins Conference 2025

How Cryo-EM Structures Reveal Molecular Mechanisms of Biomolecular Complexes (114991)

Timothy Newing 1 , Jordan Nicholls 1 2 , Jodi Brewster 1 2 , Nikolas Johnston 1 , Michael Miller 3 , Catherine Dawson 3 , Lucy Fitschen 1 2 , James Bouwer 1 2 , Simon Brown 1 , Haibo Yu 1 , Aaron Oakley 1 , Peter Lewis 1 3 , Gökhan Tolun 1
  1. School of Chemistry and Molecular Bioscience, Molecular Horizons, University of Wollongong, Wollongong, NSW, Australia
  2. ARC Industrial Transformation Training Centre for Cryo-electron Microscopy of Membrane Proteins, UOW Node, Wollongong, NSW, Australia
  3. School of Environmental and Life Sciences, University of Newcastle, Newcastle, NSW, Australia

Structures that have been elusive for decades are now being determined using Cryogenic electron microscopy (cryo-EM). Our group has determined cryo-EM structures of two RNA polymerase (RNAP) complexes from the bacterium Bacillus subtilis, and two DNA annealase complexes from a bacteriophage and a human virus. Comparison of the two RNAP structures, one with and the other without the HelD factor that removes stalled RNAP complexes from DNA, allowed us to propose a molecular mechanism for HelD’s action (1). Our phage lambda annealase structure had been sought after for more than half a century. This structure revealed the unusual DNA structure formed by this protein to enable the search for the correct base-pairing during single-strand annealing (SSA) homologous DNA recombination. It also shows how this protein forms large helical structures to accommodate long DNA molecules (2). Furthermore, this structure helped in conclusively establishing a new protein superfamily, bringing a decades-long hypothesis discussion in the field to a close, in addition to helping define a new protein fold (3). Our second annealase structure, obtained from a Human Herpes Virus, reveals entirely different mechanisms employed by these viruses to form long helical assemblies and promote DNA annealing (manuscript in prep).

  1. Newing TP, Brewster JL, Fitschen LJ, Bouwer JC, Johnston NP, Yu H, Tolun G. Redβ177 annealase structure reveals details of oligomerization and λ Red-mediated homologous DNA recombination. Nat Commun. 2022 Sep 26;13(1):5649. doi: 10.1038/s41467-022-33090-6. PMID: 36163171; PMCID: PMC9512822.
  2. Newing TP, Oakley AJ, Miller M, Dawson CJ, Brown SHJ, Bouwer JC, Tolun G, Lewis PJ. Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD. Nat Commun. 2020 Dec 18;11(1):6420. doi: 10.1038/s41467-020-20157-5. PMID: 33339820; PMCID: PMC7749167.
  3. Al-Fatlawi A, Schroeder M, Stewart AF. The Rad52 SSAP superfamily and new insight into homologous recombination. Commun Biol. 2023 Jan 23;6(1):87. doi: 10.1038/s42003-023-04476-z. PMID: 36690694; PMCID: PMC9870868.