3/11/2023 0 Comments Protein scaffold pdf![]() The RuvC domain (residues 60–253) encompasses the three catalytic motifs (RuvC I–III) and three insertions (BH (residues 92–112), A (residues 113–129) and B (residues 161–179)) (Fig. The structure revealed that IsrB extensively binds to target DNA through a 20-nt duplex between the ωRNA and target DNA (Fig. ![]() On the basis of this ωRNA model and an initial IsrB model generated by protein structure prediction, we determined the IsrB–ωRNA–DNA structure (Fig. To refine the modelling of the RNA coordinates, we used an RNA-specific modelling tool, auto-DRRAFTER, together with a covariance-based secondary structure model to build an initial ωRNA model. ![]() Some regions of the map corresponding to the ωRNA, however, were resolved at a lower resolution. We obtained a three-dimensional (3D) reconstruction of the ternary complex with an overall resolution of 3.1 Å (Fig. ![]() To characterize the molecular mechanism of ωRNA-guided DNA targeting by IsrB, we analysed a ternary complex comprising Desulfovirgula thermocuniculi IsrB (DtIsrB), a 284-nt ωRNA containing a 20-nt guide segment, a 31-nt target DNA strand and a 10-nt non-target DNA strand using single-particle cryo-EM (Fig. Previous work has shown that IsrB associates with a roughly 300-nt ωRNA, which guides IsrB to nick the non-target strand of double-stranded (ds) DNA containing a 5′-NTGA-3′ target-adjacent motif (TAM) 4. IsrB additionally contains an N-terminal PLMP domain (named after its conserved amino acid motif) and an uncharacterized C-terminal domain (Fig. However, in contrast to IscB and Cas9, IsrB lacks the HNH nuclease domain, the REC lobe and large portions of the protospacer adjacent motif- (PAM-)interacting domain and, accordingly, is much smaller (at roughly 350 amino acids) than Cas9. Like IscB and Cas9, IsrB contains a RuvC-like nuclease domain that is interrupted by the insertion of a bridge helix (BH) (Fig. IsrB is the likely antecedent of IscB, another OMEGA family member that is the apparent ancestor of Cas9, as indicated both by phylogenetic analysis and by the shared unique domain architecture 4, 5. The RNA-guided IsrB protein is an OMEGA family member encoded in the IS200/IS605 superfamily of transposons. Structural analyses of IsrB and its ωRNA as well as comparisons to other RNA-guided systems highlight the functional interplay between protein and RNA, advancing our understanding of the biology and evolution of these diverse systems. In contrast to Cas9, however, which uses a recognition (REC) lobe to facilitate target selection, IsrB relies on its ωRNA, part of which forms an intricate ternary structure positioned analogously to REC. We find the overall structure of the IsrB protein shares a common scaffold with Cas9. Here, we report the cryogenic-electron microscopy structure of Desulfovirgula thermocuniculi IsrB (DtIsrB) in complex with its cognate ωRNA and a target DNA. ![]() IsrB consists of only around 350 amino acids, but its small size is counterbalanced by a relatively large RNA guide (roughly 300-nt ωRNA). Recent work identified a new class of RNA-guided systems, termed OMEGA, which include IscB, the likely ancestor of Cas9, and the nickase IsrB, a homologue of IscB lacking the HNH nuclease domain 4. Structural studies of these systems have illuminated how the RNA and protein jointly recognize and cleave their substrates, guiding rational engineering for further technology development 3. RNA-guided systems, such as CRISPR–Cas, combine programmable substrate recognition with enzymatic function, a combination that has been used advantageously to develop powerful molecular technologies 1, 2. ![]()
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