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TitleStructures of the holo CRISPR RNA-guided transposon integration complex.
Journal, issue, pagesNature, Vol. 613, Issue 7945, Page 775-782, Year 2023
Publish dateNov 28, 2022
AuthorsJung-Un Park / Amy Wei-Lun Tsai / Alexandrea N Rizo / Vinh H Truong / Tristan X Wellner / Richard D Schargel / Elizabeth H Kellogg /
PubMed AbstractCRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR ...CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.
External linksNature / PubMed:36442503 / PubMed Central
MethodsEM (single particle)
Resolution3.0 - 3.7 Å
Structure data

25453

7svu

EMDB-25453, PDB-7svu:
TnsBctd-TnsC-TniQ complex
Method: EM (single particle) / Resolution: 3.5 Å

27971

8ea3

EMDB-27971, PDB-8ea3:
V-K CAST Transpososome from Scytonema hofmanni, major configuration
Method: EM (single particle) / Resolution: 3.7 Å

27972

8ea4

EMDB-27972, PDB-8ea4:
V-K CAST Transpososome from Scytonema hofmanni, minor configuration
Method: EM (single particle) / Resolution: 3.0 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

Source
  • [scytonema hofmanni] utex 2349 (bacteria)
  • synthetic construct (others)
  • scytonema hofmannii (bacteria)
  • escherichia coli (E. coli)
KeywordsDNA BINDING PROTEIN/DNA / CAST / transposase / AAA+ ATPase / AAA+ / CRISPR / Cas / DNA BINDING PROTEIN-DNA complex / DNA BINDING PROTEIN/DNA/RNA / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA complex / DNA BINDING PROTEIN/RNA/DNA / DNA BINDING PROTEIN-RNA-DNA complex

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