8ea3
From Proteopedia
V-K CAST Transpososome from Scytonema hofmanni, major configuration
Structural highlights
FunctionRS15_ECOLI One of the primary rRNA binding proteins, it binds directly to 16S rRNA where it helps nucleate assembly of the platform of the 30S subunit by binding and bridging several RNA helices of the 16S rRNA.[HAMAP-Rule:MF_01343] In the E.coli 70S ribosome it has been modeled (PubMed:12809609) to contact the 23S rRNA of the 50S subunit forming part of bridge B4. In the two 3.5 A resolved ribosome structures (PubMed:16272117) there are minor differences between side-chain conformations.[HAMAP-Rule:MF_01343] Publication Abstract from PubMedCRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism(1-3). 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. Structures of the holo CRISPR RNA-guided transposon integration complex.,Park JU, Tsai AW, Rizo AN, Truong VH, Wellner TX, Schargel RD, Kellogg EH Nature. 2023 Jan;613(7945):775-782. doi: 10.1038/s41586-022-05573-5. Epub 2022 , Nov 28. PMID:36442503[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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