RT Journal Article
SR Electronic
T1 Structural basis for target-site selection in RNA-guided DNA transposition systems
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.05.25.445634
DO 10.1101/2021.05.25.445634
A1 Jung-Un Park
A1 Amy Tsai
A1 Eshan Mehrotra
A1 Michael T. Petassi
A1 Shan-Chi Hsieh
A1 Ailong Ke
A1 Joseph E. Peters
A1 Elizabeth H. Kellogg
YR 2021
UL http://biorxiv.org/content/early/2021/05/25/2021.05.25.445634.abstract
AB CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA insertion in one orientation at a fixed distance from a programmable target sequence. We define the mechanism explaining this process by characterizing the transposition regulator, TnsC, from a Type V-K CRISPR-transposase system using cryo-EM. Polymerization of ATP-bound TnsC helical filaments explains how polarity information is passed to the transposase. Our Cryo-EM structure of TniQ-TnsC reveals that TniQ caps the TnsC filament, establishing 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, structures with the transition state mimic, ADP·AlF3, reveals how TnsC transitions to define the fixed point of insertion. These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.One Sentence Summary Cryo-EM studies reveals the role of the AAA+ regulator TnsC for target-site selection in CRISPR-associated transposition systems.Competing Interest StatementThe Peters lab has corporate funding for research that is not directly related to the work in this publication. Cornell University has filed patent applications with J.E.P. as inventor involving CRISPR-Cas systems associated with transposons that is not directly related to this work.