In prokaryotes, Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNAs (crRNAs), together with CRISPR-associated (Cas) proteins, capture and degrade invading genetic materials. In the type III-B CRISPR-Cas system, six Cas proteins (Cmr1-Cmr6) and a crRNA form an RNA silencing Cmr complex. Here we report the 2.1 Å crystal structure of the Cmr1-deficient, functional Cmr complex bound to single-stranded DNA, a substrate analog complementary to the crRNA guide. Cmr3 recognizes the crRNA 5' tag and defines the start position of the guide-target duplex, using its idiosyncratic loops. The β-hairpins of three Cmr4 subunits intercalate within the duplex, causing nucleotide displacements with 6 nt intervals, and thus periodically placing the scissile bonds near the crucial aspartate of Cmr4. The structure reveals the mechanism for specifying the periodic target cleavage sites from the crRNA 5' tag and provides insights into the assembly of the type III interference machineries and the evolution of the Cmr and Cascade complexes.

The 5-carboxymethylaminomethyl modification of uridine (cmnm(5)U) at the anticodon first position occurs in tRNAs that read split codon boxes ending with purine. This modification is crucial for correct translation, by restricting codon-anticodon wobbling. Two conserved enzymes, GidA and MnmE, participate in the cmnm(5)U modification process. Here we determined the crystal structure of Aquifex aeolicus GidA at 2.3 A resolution. The structure revealed the tight interaction of GidA with FAD. Structure-based mutation analyses allowed us to identify two conserved Cys residues in the vicinity of the FAD-binding site that are essential for the cmnm(5)U modification in vivo. Together with mutational analysis of MnmE, we propose a mechanism for the cmnm(5)U modification process where GidA, but not MnmE, attacks the C6 atom of uridine by a mechanism analogous to that of thymidylate synthase. We also present a tRNA-docking model that provides structural insights into the tRNA recognition mechanism for efficient modification.

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The substrate-binding mode of a 26-kDa GH19 chitinase from rye, Secale cereale, seeds (RSC-c) was investigated by crystallography, sitedirected mutagenesis and NMR spectroscopy. The crystal structure of RSC-c in a complex with an N-acetylglucosamine tetramer, (GlcNAc) 4 , was successfully solved, and revealed the binding mode of the tetramer to be an aglycon-binding site, subsites +1, +2, +3, and +4. These are the first crystallographic data showing the oligosaccharide-binding mode of a family GH19 chitinase. From HPLC analysis of the enzymatic reaction products, mutation of Trp72 to alanine was found to affect the product distribution obtained from the substrate, p-nitrophenyl penta-N-acetyl-bchitopentaoside. Mutational experiments confirmed the crystallographic finding that the Trp72 side chain interacts with the +4 moiety of the bound substrate. To further confirm the crystallographic data, binding experiments were also conducted in solution using NMR spectroscopy. Several signals in the 1 H-15 N HSQC spectrum of the stable isotope-labeled RSC-c were affected upon addition of (GlcNAc) 4 . Signal assignments revealed that most signals responsive to the addition of (GlcNAc) 4 are derived from amino acids located at the surface of the aglycon-binding site. The binding mode deduced from NMR binding experiments in solution was consistent with that from the crystal structure. DatabaseThe atomic coordinates and structural factors have been deposited in the Protein Data Bank, under the accession codes 4DWX (unliganded form) and 4DYG ((GlcNAc) 4 complex). Chitinase, EC 3.2.1.14. Backbone assignment data were deposited in the Biological Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu/bmrb) with the code number 11467Structured digital abstract l RSC-c and RSC-c bind by x-ray crystallography (View interaction) Abbreviations (GlcNAc) n , b-1,4-linked oligosaccharides of GlcNAc with a polymerization degree of n; GlcNAc, N-acetylglucosamine; pNP, p-nitrophenyl; RSC-c, a 'loopful' family GH19 chitinase from rye seeds; W72A RSC-c, RSC-c in which Trp 72 is mutated to alanine.

The cytidine at the first position of the anticodon (C34) in the AUA codon-specific archaeal tRNA(Ile2) is modified to 2-agmatinylcytidine (agm(2)C or agmatidine), an agmatine-conjugated cytidine derivative, which is crucial for the precise decoding of the genetic code. Agm(2)C is synthesized by tRNA(Ile)-agm(2)C synthetase (TiaS) in an ATP-dependent manner. Here we present the crystal structures of the Archaeoglobus fulgidus TiaS-tRNA(Ile2) complexed with ATP, or with AMPCPP and agmatine, revealing a previously unknown kinase module required for activating C34 by phosphorylation, and showing the molecular mechanism by which TiaS discriminates between tRNA(Ile2) and tRNA(Met). In the TiaS-tRNA(Ile2)-ATP complex, C34 is trapped within a pocket far away from the ATP-binding site. In the agmatine-containing crystals, C34 is located near the AMPCPP γ-phosphate in the kinase module, demonstrating that agmatine is essential for placing C34 in the active site. These observations also provide the structural dynamics for agm(2)C formation.