During translation termination, class II release factor RF3 binds to the ribosome to promote rapid dissociation of a class I release factor (RF) in a GTP-dependent manner. We present the crystal structure of E. coli RF3*GDP, which has a three-domain architecture strikingly similar to the structure of EF-Tu*GTP. Biochemical data on RF3 mutants show that a surface region involving domains II and III is important for distinct steps in the action cycle of RF3. Furthermore, we present a cryo-electron microscopy (cryo-EM) structure of the posttermination ribosome bound with RF3 in the GTP form. Our data show that RF3*GTP binding induces large conformational changes in the ribosome, which break the interactions of the class I RF with both the decoding center and the GTPase-associated center of the ribosome, apparently leading to the release of the class I RF.

During translation, the ribosome and several of its constituent proteins undergo structural transitions between different functional states. Protein L12, present in four copies in prokaryotic ribosomes, forms a flexible "stalk" with key functions in factor-dependent GTP hydrolysis during translocation. Here we have used heteronuclear NMR spectroscopy to characterize L12 conformation and dynamics in solution and on the ribosome. Isolated L12 forms a symmetric dimer mediated by the N-terminal domains (NTDs), to which each C-terminal domain (CTD) is connected via an unstructured hinge segment. The overall structure can be described as three ellipsoids joined by flexible linkers. No persistent contacts are seen between the two CTDs, or between the NTD and CTD in the L12 dimer in solution. In the (1)H-(15)N HSQC spectrum of the Escherichia coli 70S ribosome, a single set of cross-peaks are observed for residues 40-120 of L12, the intensities of which correspond to only two of four protein copies. The structure of the CTDs observed on the ribosome is indistinguishable from that of isolated L12. These results indicate that two CTDs with identical average structures are mobile and extend away from the ribosome, while the other two copies most likely interact tightly with the ribosome even in the absence of translational factors.

The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EFTu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing PhetRNA Phe , Trp-tRNA Trp , or Leu-tRNA LeuI . The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All three aa-tRNAs have the same 'loaded spring' conformation with a kink and twist between the D-stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF-Tu-binding CCA end of aa-tRNA, proposed to signal cognate codon-anticodon interaction to the GTPase centre and tune the accuracy of aa-tRNA selection.

It has been suggested from in vivo and cryoelectron micrographic studies that the large ribosomal subunit protein L11 and its N-terminal domain play an important role in peptide release by, in particular, the class I release factor RF1. In this work, we have studied in vitro the role of L11 in translation termination with ribosomes from a wild type strain (WT-L11), an L11 knocked-out strain (⌬L11), and an L11 N terminus truncated strain (Cter-L11). Our data show 4 -6-fold reductions in termination efficiency (k cat /K m ) of RF1, but not of RF2, on ⌬L11 and Cter-L11 ribosomes compared with wild type. There is, at the same time, no effect of these L11 alterations on the maximal rate of ester bond cleavage by either RF1 or RF2. The rates of dissociation of RF2 but not of RF1 from the ribosome after peptide release are somewhat reduced by the L11 changes irrespective of the presence of RF3, and they cause a 2-fold decrease in the missense error. Our results suggest that the L11 modifications increase nonsense suppression at UAG codons because of the reduced termination efficiency of RF1 and that they decrease nonsense suppression at UGA codons because of a decreased missense error level.L11 is a highly conserved ribosomal 14.8-kDa protein located at the base of the L7/L12 stalk of the ribosome, which is essential for several steps in protein synthesis (1-5). L11 binds to the nucleotides 1051-1108 of Escherichia coli 23 S rRNA, commonly called the L11 binding region (L11BR) 2 (6), which constitutes the GTPase-associated center, an important sector of the bacterial ribosome, where all of the translational GTPases bind and hydrolyze GTP in the course of their action (7). This is also the site of action for the thiazole antibiotics thiostrepton and micrococcin (8 -10). In addition to the GTPases, some other translational factors interact with L11 and the L11BR in functionally important ways. The class I release factors RF1 and RF2 belong to this group. RF1 recognizes the stop codons UAG and UAA, whereas RF2 recognizes the stop codons UGA and UAA in the A site of the ribosome (11). Recent cryo-EM studies with termination complexes containing RF2 (12, 13) and RF1 3 show that these two factors acquire very similar overall conformations on the ribosome. Although they bind to the decoding center on the 30 S subunit and reach up to the peptidyltransferase center on the 50 S subunit to induce release of the nascent peptide chain, they interact with L11BR (12, 13) and L11 3 with their flexible domain I. These observations are in line with previous suggestions, based on biochemical and genetic experiments, that there are interactions between L11 and the release factors (4, 14 -18).The L11 protein consists of two domains, a tightly folded N-terminal domain (NTD), which is loosely connected to the large compact C-terminal domain (CTD). The CTD of L11 is in stable contact with the L11BR RNA, whereas the NTD can change its position and proximity with respect to the rest of the protein (19). Earlier biochemical and genetic studies i...