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...

Twister is a recently discovered RNA motif that is estimated to have one of the fastest known catalytic rates of any naturally occurring small self-cleaving ribozyme. We determined the 4.1-Å resolution crystal structure of a twister sequence from an organism that has not been cultured in isolation, and it shows an ordered scissile phosphate and nucleotide 5′ to the cleavage site. A second crystal structure of twister from Orzyza sativa determined at 3.1-Å resolution exhibits a disordered scissile phosphate and nucleotide 5′ to the cleavage site. The core of twister is stabilized by base pairing, a large network of stacking interactions, and two pseudoknots. We observe three nucleotides that appear to mediate catalysis: a guanosine that we propose deprotonates the 2′-hydroxyl of the nucleotide 5′ to the cleavage site and a conserved adenosine. We suggest the adenosine neutralizes the negative charge on a nonbridging phosphate oxygen atom at the cleavage site. The active site also positions the labile linkage for in-line nucleophilic attack, and thus twister appears to simultaneously use three strategies proposed for small self-cleaving ribozymes. The twister crystal structures (i) show its global structure, (ii) demonstrate the significance of the double pseudoknot fold, (iii) provide a possible hypothesis for enhanced catalysis, and (iv) illuminate the roles of all 10 highly conserved nucleotides of twister that participate in the formation of its small and stable catalytic pocket.X-ray crystallography | RNA structure | weak derivative phasing | samarium derivatives | cesium derivatives T he twister RNA motif was identified by bioinformatic searches and then validated biochemically to be a small self-cleaving ribozyme (1). This recently discovered class of ribozymes is called twister because its conserved secondary structure resembles the ancient Egyptian hieroglyph "twisted flax." Representatives of the twister ribozyme class are found in all domains of life, but its biological role has yet to be determined. In addition to twister, the small self-cleaving ribozyme family includes the hammerhead, hairpin, hepatitis delta virus (HDV), Varkud satellite (VS), and glmS ribozymes (the glmS ribozyme is upsteam of the the glmS gene that codes for the enzyme that catalyzes glucosamine-6-phosphate production) (1-6).The small self-cleaving ribozyme family can be split into two groups based on whether their active site is formed by an irregular helix (hammerhead, hairpin, and VS) or a double pseudoknot (PK) structure (HDV and glmS) (7). The structures of HDV and glmS are known, whereas twister was predicted from representative sequences to use two PKs to form its active site. It was expected that twister would be smaller in size than either HDV or glmS and more comparable in size and complexity to hammerhead (1).The self-cleavage rate constant of twister is estimated to be as rapid as or slightly more rapid than the hammerhead ribozyme. The estimated rate constants (k obs ) for twister is 1,000 per minute, and the experime...

Translation initiation factor 2 (IF2) promotes 30S initiation complex (IC) formation and 50S subunit joining, which produces the 70S IC. The architecture of full-length IF2, determined by small angle X-ray diffraction and cryo electron microscopy, reveals a more extended conformation of IF2 in solution and on the ribosome than in the crystal. The N-terminal domain is only partially visible in the 30S IC, but in the 70S IC, it stabilizes interactions between IF2 and the L7/ L12 stalk of the 50S, and on its deletion, proper N-formyl-methionyl (fMet)-tRNA fMet positioning and efficient transpeptidation are affected. Accordingly, fast kinetics and single-molecule fluorescence data indicate that the N terminus promotes 70S IC formation by stabilizing the productive sampling of the 50S subunit during 30S IC joining. Together, our data highlight the dynamics of IF2-dependent ribosomal subunit joining and the role played by the N terminus of IF2 in this process.protein synthesis | integrated structural biology I n bacteria, three translation initiation factors (IF1, IF2, and IF3) participate in the first phase of protein synthesis during which the 30S subunit binds the initiator tRNA to form codonanticodon interactions with the mRNA. The 30S initiation complex (IC) thus formed is converted into a 70S IC by joining of the 50S ribosomal subunit (1, 2). The IFs enhance the rate of ICs formation and ensure translation accuracy. Within this process, IF2 plays a key role by promoting the recruitment and stabilization of the N-formyl-methionyl(fMet)-tRNA fMet on the 30S IC in the peptidyl/initiator (P/I) site of the small ribosomal subunit with the help of initiation factors IF1 and IF3 (3-6). IF2 mediates subunit joining on which its GTPase activity is stimulated, leading to the formation of a 70S IC in a conformation that is productive in the first transpeptidation reaction (7). Snapshots of this process, visualized by cryo electron microscopy (cryo-EM) (3,(8)(9)(10), have localized IF2 on the ribosome and provided structural insights into 30S and 70S IC's.Bacterial IF2 is a multidomain protein (Figs. S1 and S2) for which the overall architecture is unknown. Unlike 70S-interacting elongation GTPases, IF2 contains a specific N-terminal (N) domain that is variable in sequence (in Escherichia coli, two copies of it comprise the bona fide N domain and the G1 domain). Furthermore, it contains a highly conserved C-terminal region with a central core comprising domains G [GTP/GDP (G) binding domain, G2] and G3 (II), and a C-terminal part consisting of domain C1 and of the initiator tRNA-binding domain C2. Recently, the crystal structure of the core part (1-363) of Thermus thermophilus IF2 was determined in different functional states (apo-protein, GTP, and GDP forms) (11). These structures gave important insights into the mechanism of nucleotide binding and GTP hydrolysis and also reveal significant structural differences with a related archaeal and eukaryotic protein a/eIF5B (11). However, because the entire C-terminal region was ...

The initiation of protein synthesis uses initiation factor 2 (IF2) in prokaryotes and a related protein named eukaryotic initiation factor 5B (eIF5B) in eukaryotes. IF2 is a GTPase that positions the initiator tRNA on the 30S ribosomal initiation complex and stimulates its assembly to the 50S ribosomal subunit to make the 70S ribosome. The 3.1-Å resolution X-ray crystal structures of the fulllength Thermus thermophilus apo IF2 and its complex with GDP presented here exhibit two different conformations (all of its domains except C2 domain are visible). Unlike all other translational GTPases, IF2 does not have an effecter domain that stably contacts the switch II region of the GTPase domain. The domain organization of IF2 is inconsistent with the "articulated lever" mechanism of communication between the GTPase and initiator tRNA binding domains that has been proposed for eIF5B. Previous cryo-electron microscopy reconstructions, NMR experiments, and this structure show that IF2 transitions from being flexible in solution to an extended conformation when interacting with ribosomal complexes.T he synthesis of proteins in prokaryotes is divided into three distinct processes: initiation, elongation, and termination. The initiation of translation in prokaryotes is directed by three initiation factors (IF1, IF2, and IF3) that govern the binding and positioning of the mRNA, as well as the initiator tRNA, and the joining of ribosomal subunits to form a 70S complex that is ready for the elongation stage of protein synthesis.IF2 is a GTPase that functions to position the initiator tRNA within the 30S ribosomal initiation complex (30S IC) and promotes its joining with the 50S ribosomal subunit to form a 70S ribosome. IF2 is encoded by a single copy of the infB gene and is completely conserved in bacteria (1). The flexible structure of the N terminus has the largest sequence variability among different species (2). Variability also exists within a species; for instance, Escherichia coli IF2 has three isoforms, which vary in the length of their N-terminal domain due to three distinct start sites for its translation initiation (1). The C-terminal part of IF2 (G, II, C1, and C2 domains) contains the highly conserved GTPase domain (G domain) and C2 domain, which interact with the initiator tRNA.The C2 domain recognizes and protects the formylated Met of the initiator tRNA from hydrolysis (3, 4). The formylation of Met results in a fivefold increase in the binding affinity of the tRNA and is made in a G-nucleotide-independent fashion (3, 5). This interaction permits IF2 to assist in positioning the initiator tRNA within a 30S initiation complex and guide the formation of a functional 30S IC on the establishment of the P-site codonanticodon interaction (2, 6, 7). A functional 30S IC is competent for the 50S ribosomal subunit to join, which is mediated by the formation of the intersubunit salt bridges via an interaction between IF2 and L12 (8). Once the 50S ribosomal subunit joins, IF2 comes into contact with the GTPase activation c...

Histidine transfer RNA (tRNA) is unique among tRNA species as it carries an additional nucleotide at its 5′ terminus. This unusual G−1 residue is the major tRNAHis identity element, and essential for recognition by the cognate histidyl-tRNA synthetase to allow efficient His-tRNAHis formation. In many organisms G−1 is added post-transcriptionally as part of the tRNA maturation process. tRNAHis guanylyltransferase (Thg1) specifically adds the guanylyate residue by recognizing the tRNAHis anticodon. Thg1 homologs from all three domains of life have been the subject of exciting research that gave rise to a detailed biochemical, structural and phylogenetic enzyme characterization. Thg1 homologs are phylogenetically classified into eukaryal- and archaeal-type enzymes differing characteristically in their cofactor requirements and specificity. Yeast Thg1 displays a unique but limited ability to add 2–3 G or C residues to mutant tRNA substrates, thus catalyzing a 3′ → 5′ RNA polymerization. Archaeal-type Thg1, which has been horizontally transferred to certain bacteria and few eukarya, displays a more relaxed substrate range and may play additional roles in tRNA editing and repair. The crystal structure of human Thg1 revealed a fascinating structural similarity to 5′ → 3′ polymerases, indicating that Thg1 derives from classical polymerases and evolved to assume its specific function in tRNAHis processing.