During peptide bond formation on the ribosome the α-amine of an aminoacyl-tRNA attacks the ester carbonyl carbon of a peptidyl-tRNA to yield a peptide lengthened by one amino acid. Although the ribosome's contribution to catalysis is predominantly entropic, the lack of high-resolution structural data for the complete active site in complex with full-length ligands has made it difficult to assess how the ribosome might influence the pathway of the reaction. Here, we present crystal structures of pre-attack and post-catalysis complexes of the Thermus thermophilus 70S ribosome at ∼2.6 Å resolution. These structures reveal a network of hydrogen bonds along which proton transfer could take place to ensure the concerted, rate-limiting formation of a tetrahedral intermediate. Unlike earlier models, we propose that the ribosome and the A-site tRNA facilitate the deprotonation of the nucleophile through the activation of a water molecule.

Nucleosomes uniquely positioned on high-affinity DNA sequences present a polar barrier to transcription by human and yeast RNA polymerase II (Pol II). In one transcriptional orientation, these nucleosomes provide a strong, factor- and salt-insensitive barrier at the entry into the H3/H4 tetramer that can be recapitulated without H2A/H2B dimers. The same nucleosomes transcribed in the opposite orientation form a weaker, more diffuse barrier that is largely relieved by higher salt, TFIIS, or FACT. Barrier properties are therefore dictated by both the local nucleosome structure (influenced by the strength of the histone-DNA interactions) and the location of the high-affinity DNA region within the nucleosome. Pol II transcribes DNA sequences at the entry into the tetramer much less efficiently than the same sequences located distal to the nucleosome dyad. Thus, entry into the tetramer by Pol II facilitates further transcription, perhaps due to partial unfolding of the tetramer from DNA.

Here we report the crystal structures of the Thermus thermophilus ribosome at 2.3-2.5Å-resolution, which have enabled a comprehensive modeling of rRNA modifications. The structures reveal contacts of modified nucleotides with mRNA and tRNAs or protein pY, and contacts within the ribosome interior stabilizing the functional fold of rRNA. Our work provides a resource to explore the roles of rRNA modifications and yields the most complete atomic model of bacterial ribosome.