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Structure paper

TitleStructural basis for target site selection in RNA-guided DNA transposition systems.
Journal, issue, pagesScience, Vol. 373, Issue 6556, Page 768-774, Year 2021
Publish dateAug 13, 2021
AuthorsJung-Un Park / Amy Wei-Lun Tsai / Eshan Mehrotra / Michael T Petassi / Shan-Chi Hsieh / Ailong Ke / Joseph E Peters / Elizabeth H Kellogg /
PubMed AbstractCRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron ...CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate-bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.
External linksScience / PubMed:34385391 / PubMed Central
MethodsEM (helical sym.) / EM (single particle)
Resolution3.2 - 4.2 Å
Structure data

23720

7m99

EMDB-23720, PDB-7m99:
ATPgS bound TnsC filament from ShCAST system
Method: EM (helical sym.) / Resolution: 3.2 Å

23721

7m9a

EMDB-23721, PDB-7m9a:
ADP-AlF3 bound TnsC structure from ShCAST system
Method: EM (single particle) / Resolution: 3.9 Å

23722

7m9b

EMDB-23722, PDB-7m9b:
ADP-AlF3 bound TnsC structure in closed form
Method: EM (single particle) / Resolution: 3.8 Å

23723

7m9c

EMDB-23723, PDB-7m9c:
ADP-AlF3 bound TnsC structure in open form
Method: EM (single particle) / Resolution: 4.2 Å

EMDB-23724:
ATP bound TnsC filament structure from ShCAST system
Method: EM (helical sym.) / Resolution: 3.7 Å

EMDB-23725:
Head-to-head filament of TnsC from ShCAST system
Method: EM (single particle) / Resolution: 4.2 Å

23726

7n6i

EMDB-23726, PDB-7n6i:
ATP-bound TnsC-TniQ complex from ShCAST system
Method: EM (single particle) / Resolution: 3.9 Å

Chemicals

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

ChemComp-MG:
Unknown entry

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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

Source
  • scytonema hofmannii (bacteria)
  • synthetic construct (others)
KeywordsDNA BINDING PROTEIN/DNA / CRISPR / Transposition / AAA+ ATPase / Tn7 / DNA BINDING PROTEIN-DNA complex / DNA BINDING PROTEIN / Transposase

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