One-sentence summary: A series of X-ray crystal structures of bacterial Glu-tRNA Gln -dependent amidotransferase (GatCAB) in the apo form and in the substrate-bound states, combined with the biochemical studies, elucidate the sophisticated mechanism to synthesize Gln-tRNA Gln , coupling the glutaminase with the kinase and transamidase reactions by a characteristic ammonia channel 30 Å in length.

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA Pyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N e -acetyl-Lys (AcK) onto tRNA Pyl . Here, we examine an N e -acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.aminoacyl-tRNA synthetase | genetic code | genetic selection | posttranslational modification | synthetic biology T he standard genetic code table relates the 64 nucleotide triplets to three stop signals and 20 canonical amino acids. Some organisms, including humans, naturally evolved expanded genetic codes that accommodate 21 amino acids (1), or possibly 22 amino acids in rare cases (2). Engineering translation system components, including tRNAs (3, 4), aminoacyl-tRNA synthetases (AARSs) (5, 6), elongation factors (7), and the ribosome itself (8), have produced organisms with artificially expanded genetic codes. Products of genetic code engineering include bacterial, yeast, and mammalian cells and animals that are able to synthesize proteins with sitespecifically inserted noncanonical amino acids (ncAAs) (9).Genetic code expansion systems rely on an orthogonal AARS/ tRNA pair (o-AARS, o-tRNA) (5, 6). The o-AARS should be specific in ligating a desired ncAA to a stop codon decoding tRNA, and both the o-tRNA and o-AARS are assumed not to cross-react with endogenous AARSs or tRNAs. Although some AARSs evolved in nature to recognize certain ncAAs (10-12), many genetic code expansion systems require a mutated AARS active site. The active site of the o-AARS is usually redesigned via directed evolution (6), including positive and negative selective rounds, to produce an enzyme that is assumed to be specific for an ncAA and not active with the 20 canonical amino acids. Genetic code expansion technology is rapidly evolving (13), and the ability to incorporate multiple ncAAs into a protein using quadruplet-codon decoding (14) or sense-codon recoding (15-19) is now becoming feasible. Protein synthesis with multiple ncAAs will require o-AARSs that are able to discriminate their ncAA substrate not only from canonical amino acids in the cell but from other ncAAs that are added to the cell.Probing the effects of amino acid analogs on bacterial cell...

The RtcB protein has recently been identified as a 3′-phosphate RNA ligase that directly joins an RNA strand ending with a 2′,3′-cyclic phosphate to the 5′-hydroxyl group of another RNA strand in a GTP/Mn 2+ -dependent reaction. Here, we report two crystal structures of Pyrococcus horikoshii RNA-splicing ligase RtcB in complex with Mn 2+ alone (RtcB/ Mn 2+) and together with a covalently bound GMP (RtcB-GMP/Mn 2+ ). The RtcB/ Mn 2+ structure (at 1.6 Å resolution) shows two Mn 2+ ions at the active site, and an array of sulfate ions nearby that indicate the binding sites of the RNA phosphate backbone. The structure of the RtcB-GMP/Mn 2+ complex (at 2.3 Å resolution) reveals the detailed geometry of guanylylation of histidine 404. The critical roles of the key residues involved in the binding of the two Mn 2+ ions, the four sulfates, and GMP are validated in extensive mutagenesis and biochemical experiments, which also provide a thorough characterization for the three steps of the RtcB ligation pathway: (i) guanylylation of the enzyme, (ii) guanylyl-transfer to the RNA substrate, and (iii) overall ligation. These results demonstrate that the enzyme's substrate-induced GTP binding site and the putative reactive RNA ends are in the vicinity of the binuclear Mn 2+ active center, which provides detailed insight into how the enzyme-bound GMP is tansferred to the 3′-phosphate of the RNA substrate for activation and subsequent nucleophilic attack by the 5′-hydroxyl of the second RNA substrate, resulting in the ligated product and release of GMP.RNA repair | tRNA splicing | two-metal-ion catalysis R NA ligases join two RNA strands whose ends are produced by specific RNases in many biological processes during tRNA processing/splicing, antiphage, or unfolded protein response (1-3). Most widely studied are the 5′-Phosphate (5′-P) RNA ligases (4-6) that specifically catalyze the nucleophilic attack of a free 3′-hydroxyl on an activated 5′-P. However, these ligases cannot directly join two RNA strands ending with a 2′,3′-cyclic phosphate (RNA>p) and with a 5′-hydroxyl group. Before ligation, these strands require the action of a polynucleotide kinase (forming a 5′-P) and a phosphoesterase (generating a free 3′-OH) (6). The joining mechanism of 5′-P polynucleotide ligases involves a 3′-hydroxyl and an activated 5′-P end for internucleotide phosphodiester bond formation (7,8). The 5′-P activation enzymes include group I and II self-splicing introns, the spliceosomal apparatus, and DNA/RNA polymerases, as well as other nucleotidyl transferases (9-11). Similarly, the tRNA His guanylyltransferase ligates the 3′-OH of GTP to the 5′-terminus of tRNA through its adenylylation-activated 5′-P; this gives the appearance of reverse polarity of nucleotide addition relative to normal RNA polymerases (12).The 3′-Phosphate (3′-P) RNA ligase activities were identified three decades ago; they use a 3′-P as a donor for joining two RNAs without involvement of phosphorylation of the 5′-hydroxyl or dephosphorylation of the 2′,3′-cyclic phosphate (1...

Dihydrouridine (D) is a highly conserved modified base found in tRNAs from all domains of life. Dihydrouridine synthase (Dus) catalyzes the D formation of tRNA through reduction of uracil base with flavin mononucleotide (FMN) as a cofactor. Here, we report the crystal structures of Thermus thermophilus Dus (TthDus), which is responsible for D formation at positions 20 and 20a, in complex with tRNA and with a short fragment of tRNA (D-loop). Dus interacts extensively with the D-arm and recognizes the elbow region composed of the kissing loop interaction between T-and D-loops in tRNA, pulling U20 into the catalytic center for reduction. Although distortion of the D-loop structure was observed upon binding of Dus to tRNA, the canonical D-loop/T-loop interaction was maintained. These results were consistent with the observation that Dus preferentially recognizes modified rather than unmodified tRNAs, indicating that Dus introduces D20 by monitoring the complete L-shaped structure of tRNAs. In the active site, U20 is stacked on the isoalloxazine ring of FMN, and C5 of the U20 uracil ring is covalently cross linked to the thiol group of Cys93, implying a catalytic mechanism of D20 formation. In addition, the involvement of a cofactor molecule in uracil ring recognition was proposed. Based on a series of mutation analyses, we propose a molecular basis of tRNA recognition and D formation catalyzed by Dus.protein-tRNA complex | RNA modification | substrate recognition | X-ray crystallography