Protein synthesis is catalyzed in the peptidyl transferase center (PTC), located in the large (50S) subunit of the ribosome. No high-resolution structure of the intact ribosome has contained a complete active site including both A- and P-site tRNAs. Additionally, though structures of the 50S subunit found no ordered proteins at the PTC, biochemical evidence suggests specific proteins are capable of interacting with the 3′ ends of the tRNA ligands. Here we present structures at 3.5 Å and 3.55 Å resolution of the 70S ribosome in complex with A- and P-site tRNAs that mimic pre- and post-peptidyl transfer states. These structures demonstrate that the PTC is very similar between the 50S subunit and the intact ribosome. Additionally they reveal interactions between ribosomal proteins L16 and L27 and the tRNA substrates, helping to elucidate the role of these proteins in peptidyl transfer.

We report the crystal structure of release factor 2 bound to ribosome with an aminoacyl tRNA substrate analog at the ribosomal P site, at 3.1 Å resolution. The structure shows that upon stopcodon recognition, the universally conserved GGQ motif packs tightly into the peptidyl transferase center. Nucleotide A2602 of 23S rRNA, implicated in peptide release, packs with the GGQ motif in release factor 2. The ribose of A76 of the peptidyl-tRNA adopts the C2′-endo conformation, and the 2′ hydroxyl of A76 is within hydrogen-bond distance of the 2′ hydroxyl of A2451. The structure suggests how a catalytic water can be coordinated in the peptidyl transferase center and, together with previous biochemical and computational data, suggests a model for how the ester bond between the peptidyl tRNA and the nascent peptide is hydrolyzed.ribosome structure | translational termination | X-ray crystallography T he ribosome translates the genetic information present in mRNA into proteins (1). A crucial step of this process is the termination of protein synthesis, which involves the cleavage and release of the nascent peptide chain from the P-site tRNA when the end of the coding sequence is reached. Translational termination by the ribosome is a precise and complex step that occurs when a stop codon of an mRNA is encountered in the A site of the small ribosomal subunit. The stop codons are recognized by proteins called class I release factors (RFs). In bacteria, two class I RFs recognize the three stop codons with overlapping specificity: RF1 recognizes UAG and RF2 recognizes UGA, whereas both factors recognize UAA (2). Upon stop-codon recognition, the class I RF promotes the hydrolysis of the ester bond between the nascent polypeptide and the peptidyl tRNA at the ribosomal P site, leading to the release of the nascent polypeptide and the termination of protein synthesis (3). Eukaryotes and archaea possess a single "omnipotent" class I RF, eRF1 or aRF1, respectively, which recognizes all three stop codons (4, 5). eRF1 and aRF1 are highly homologous with each other and were proposed to have evolved independently from their bacterial counterpart (4).A major advance in our understanding in the specificity of stop-codon recognition and peptide release at the molecular level was achieved when three high-resolution crystal structures of the 70S ribosome complexed with class I RFs and their cognate stop codons were solved at atomic resolution (6-8). An analysis of the interactions of RF1 and RF2 with the decoding center in these structures provides explanations for the specificity of stop-codon recognition by these factors. However, because all three structures represent the product state with a deacylated tRNA in the P site, the mechanism of peptide release is less clear.A universally conserved GGQ motif (9) that was shown to be required for catalytic activity (10, 11) was indeed found in the peptidyl transferase center (PTC) in earlier structures by cryoEM (12, 13) or crystallography at ∼6 Å (14). Mutations introduced at the first two co...

4-, 5- and 6-Nitroindole have been investigated and compared with 3-nitropyrrole as universal bases in oligodeoxynucleotides. Of these the 5-nitroindole derivative was found to be superior giving higher duplex stability, and behaving indiscriminately towards each of the four natural bases in duplex melting experiments. 3-Nitropyrrole, whilst not discriminating between the natural bases, was found to lead to considerable destabilisation of the duplexes, particularly when multiple substitutions were made, in contrast to the 5-nitroindole nucleoside.