Oral Presentation 50th Lorne Proteins Conference 2025

Phage anti-CRISPR control by a DNA- and RNA-binding helix–turn–helix protein (116390)

Nils Birkholz 1 2 3 4 , Kotaro Kamata 1 2 , Maximilian Feussner 5 , Max E Wilkinson 6 7 8 9 10 , Christian Cuba Samaniego 11 , Angela Migur 12 , Dari Kimanius 13 14 , Marijn Ceelen 1 , Sam C Went 15 , Ben Usher 15 , Tim R Blower 15 , Chris M Brown 16 , Chase L Beisel 12 17 , Zasha Weinberg 5 , Robert D Fagerlund 1 2 3 4 , Simon A Jackson 1 2 3 4 , Peter C Fineran 1 2 3 4
  1. Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
  2. Bioprotection Aotearoa, Dunedin, New Zealand
  3. Genetics Otago, Dunedin, New Zealand
  4. Maurice Wilkins Centre for Molecular Biodiscovery, Auckland, New Zealand
  5. Bioinformatics Group, Department of Computer Science and Interdisciplinary Centre for Bioinformatics, Leipzig University, Leipzig, Germany
  6. Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
  7. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA
  8. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
  9. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
  10. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
  11. Computational Biology Department, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA
  12. Helmholtz Institute for RNA-based Infection Research, Würzburg, Germany
  13. MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
  14. CZ Imaging Institute, Redwood City, California, USA
  15. Department of Biosciences, Durham University, Durham, United Kingdom
  16. Department of Biochemistry, University of Otago, Dunedin, New Zealand
  17. Medical Faculty, University of Würzburg, Würzburg, Germany

In all organisms, regulation of gene expression must be adjusted to meet cellular requirements, which frequently involves helix–turn–helix (HTH) domain proteins. One example for HTH-mediated gene regulation is found in the arms race between bacteria and their viruses, bacteriophages (phages). When bacteria are infected by phages, they use adaptive CRISPR–Cas defence systems for protection; in turn, phages rapidly produce anti-CRISPR (Acr) proteins to inactive CRISPR–Cas. Acr proteins are frequently co-encoded in operons with HTH-containing anti-CRISPR-associated (Aca) proteins, which we and others have demonstrated to transcriptionally auto-repress acr–aca operons by promoter binding.1–4 This Aca-mediated repression after an initial burst of Acr production likely reduces fitness costs from excessive expression. However, it is unclear how a single HTH regulator can adjust anti-CRISPR production to cope with the increasing number of phage genome copies and accumulating ­acr–aca mRNAs. Here, we show that the HTH domain of the regulator Aca2 not only represses Acr synthesis transcriptionally through DNA binding, but also inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access.5 The cryogenic electron microscopy structure of the ~40-kDa Aca2–RNA complex demonstrates how the remarkably versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR–Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain proteins, it is anticipated that many more elicit regulatory control by dual DNA and RNA binding.

  1. Birkholz N, Fagerlund RD, Smith LM, Jackson SA, Fineran PC (2019) The autoregulator Aca2 mediates anti-CRISPR repression. Nucleic Acids Research 47:9658-65
  2. Stanley SY, Borges AL, Chen KH, Swaney DL, Krogan NJ, Bondy-Denomy J, Davidson AR (2019) Anti-CRISPR-associated proteins are crucial repressors of anti-CRISPR transcription. Cell 178:1452-64
  3. Shehreen S, Birkholz N, Fineran PC, Brown CM (2022) Widespread repression of anti-CRISPR production by anti-CRISPR-associated proteins. Nucleic Acids Research 50:8615-25
  4. Lee SY, Birkholz N, Fineran PC, Park HH (2022) Molecular basis of anti-CRISPR operon repression by Aca10. Nucleic Acids Research 50:8919-28
  5. Birkholz N, Kamata K, Feussner M, Wilkinson ME, Cuba Samaniego C, Migur A, Kimanius D, Ceelen M, Went SC, Usher B, Blower TR, Brown CM, Beisel CL, Weinberg Z, Fagerlund RD, Jackson SA, Fineran PC (2024) Phage anti-CRISPR control by an RNA- and DNA-binding helix–turn–helix protein. Nature 631:670-7