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.

Post-transcriptional RNA modifications in the anticodon of transfer RNAs frequently contribute to the high fidelity of protein synthesis. In eubacteria, two genome-encoded transfer RNA (tRNA) species bear the same CAU sequence as the anticodons, which are differentiated by modified cytidines at the wobble positions. The elongator tRNA Met accepts an acetyl moiety at the wobble base to form N 4 -acetylcytidine (ac 4 C): an inherent modification ensures precise decoding of the AUG codon by strengthening CÀG base-pair interaction and concurrently preventing misreading of the near cognate AUA codon. We have determined the crystal structure of tRNA Met cytidine acetyltransferase (TmcA) from Escherichia coli complexed with two natural ligands, acetyl-CoA and ADP, at 2.35 Å resolution. The structure unexpectedly reveals an idiosyncratic RNA helicase module fused with a GCN5-related N-acetyltransferase (GNAT) fold, which intimately crossinteract. Taken together with the biochemical evidence, we further unravelled the function of acetyl-CoA as an enzyme-activating switch, and propose that an RNA helicase motor driven by ATP hydrolysis is used to deliver the wobble base to the active centre of the GNAT domain.

The secondary structures of metazoan mitochondrial (mt) tRNAs Ser deviate markedly from the paradigm of the canonical cloverleaf structure; particularly, tRNA Ser GCU corresponding to the AGY codon (Y ¼ U and C) is highly truncated and intrinsically missing the entire dihydrouridine arm. None of the mt serine isoacceptors possesses the elongated variable arm, which is the universal landmark for recognition by seryl-tRNA synthetase (SerRS). Here, we report the crystal structure of mammalian mt SerRS from Bos taurus in complex with seryl adenylate at an atomic resolution of 1.65 Å . Coupling structural information with a tRNA-docking model and the mutagenesis studies, we have unraveled the key elements that establish tRNA binding specificity, differ from all other known bacterial and eukaryotic systems, are the characteristic extensions in both extremities, as well as a few basic residues residing in the amino-terminal helical arm of mt SerRS. Our data further uncover an unprecedented mechanism of a dual-mode recognition employed to discriminate two distinct 'bizarre' mt tRNAsSer by alternative combination of interaction sites.