Application of Intramolecular O-to-N Phosphoryl Transfer Reaction to Design Fluorogenic Probes to Detect Activities of Enzymes That Metabolize Short Peptides and Acylamino Acids.

We propose the design strategy of fluorogenic probes of proteases/peptidases and acylamino acid hydrolases utilizing an intramolecular O-to-N phosphoryl transfer reaction, in which the main chain of peptides or amino acids is retained from the natural substrate but the side chain was designed to attach the fluorophore. The strategy is useful to design fluorogenic probes for peptidases/proteases that do not prefer the main chain modification and acylamino acid hydrolases. We have developed the fluorogenic substrates for GGT5, GGCT, and PM20D1 and have performed the screening of PM20D1 inhibitors/activators to characterize the compounds that modify the activity of PM20D1.

Structural mechanism of bridge RNA-guided recombination.

Insertion sequence (IS) elements are the simplest autonomous transposable elements found in prokaryotic genomes. We recently discovered that IS110 family elements encode a recombinase and a non-coding bridge RNA (bRNA) that confers modular specificity for target DNA and donor DNA through two programmable loops. Here we report the cryo-electron microscopy structures of the IS110 recombinase in complex with its bRNA, target DNA and donor DNA in three different stages of the recombination reaction cycle.

Structure of the IscB-ωRNA ribonucleoprotein complex, the likely ancestor of CRISPR-Cas9.

Transposon-encoded IscB family proteins are RNA-guided nucleases in the OMEGA (obligate mobile element-guided activity) system, and likely ancestors of the RNA-guided nuclease Cas9 in the type II CRISPR-Cas adaptive immune system. IscB associates with its cognate ωRNA to form a ribonucleoprotein complex that cleaves double-stranded DNA targets complementary to an ωRNA guide segment. Although IscB shares the RuvC and HNH endonuclease domains with Cas9, it is much smaller than Cas9, mainly due to the lack of the α-helical nucleic-acid recognition lobe.

Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex.

The type III-E CRISPR-Cas effector Cas7-11, with dual RNase activities for precursor CRISPR RNA (pre-crRNA) processing and crRNA-guided target RNA cleavage, is a new platform for bacterial and mammalian RNA targeting. We report the 2.5-Å resolution cryoelectron microscopy structure of Cas7-11 in complex with a crRNA and its target RNA.