Petassi for help with transposition assays. We additionally thank Phillip Milner for help with synthesizing GO, Tristan Wellner for optimizing GO grid fabrication, Fang Zhang for help with the generation of DNA substrates, Shan-Chi “Popo” Hsieh for generously allowing us to include his alignment of V-K CAST TnsB sequences, and Michael T. We acknowledge the Extreme Science and Engineering Discovery Environment for computational resources used for image processing (MCB200090 to E.H.K.). We thank the Cornell Center for Materials Research facility, as well as Katherine Spoth and Mariena Silvestry-Ramos, for maintenance of the electron microscopes used for this research (NSF-DMR1719875). The structural information presented here will help guide future work in modifying these important systems as programmable gene integration tools. Unlike full-length TnsB, C-terminal fragments do not appear to stimulate filament disassembly using two different assays, suggesting that additional interactions between TnsB and TnsC are required for redistributing TnsC to appropriate targets. The C-terminal end of TnsB adopts a short, structured 15-residue “hook” that decorates TnsC filaments. Although not observed in the TnsB strand-transfer complex, the C-terminal end of TnsB serves a crucial role in transposase recruitment to the target site. We observe that melting of the 5′ nontransferred strand of the transposon end is a structural feature stabilized by TnsB and furthermore is crucial for donor–DNA integration. We describe the base-specific interactions along the TnsB binding site, which explain how different CAST elements can function on cognate mobile elements independent of one another. However, key structural differences in the C-terminal domains indicate that TnsB’s tetrameric architecture is stabilized by a different set of protein–protein interactions compared with MuA. Our structure of TnsB is a tetramer, revealing strong mechanistic relationships with the overall architecture of RNaseH transposases/integrases in general, and in particular the MuA transposase from bacteriophage Mu. Using cryo-electron microscopy (cryo-EM) structure determination, we reveal the conformation of TnsB during transposon integration for the type V-K CAST system from Scytonema hofmanni (ShCAST). Although structural data are known for nearly all core transposition components, the transposase component, TnsB, remains uncharacterized. CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration.
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