During the ribosomal translocation, the binding of elongation factor G (EF-G) to the pretranslocational ribosome leads to a ratchet-like rotation of the 30S subunit relative to the 50S subunit in the direction of the mRNA movement. By means of cryo-electron microscopy we observe that this rotation is accompanied by a 20 A movement of the L1 stalk of the 50S subunit, implying that this region is involved in the translocation of deacylated tRNAs from the P to the E site. These ribosomal motions can occur only when the P-site tRNA is deacylated. Prior to peptidyl-transfer to the A-site tRNA or peptide removal, the presence of the charged P-site tRNA locks the ribosome and prohibits both of these motions.

Aminoacyl-tRNAs (aa-tRNAs) are delivered to the ribosome as part of the ternary complex of aa-tRNA, elongation factor Tu (EF-Tu) and GTP. Here, we present a cryo-electron microscopy (cryo-EM) study, at a resolution of approximately 9 A, showing that during the incorporation of the aa-tRNA into the 70S ribosome of Escherichia coli, the flexibility of aa-tRNA allows the initial codon recognition and its accommodation into the ribosomal A site. In addition, a conformational change observed in the GTPase-associated center (GAC) of the ribosomal 50S subunit may provide the mechanism by which the ribosome promotes a relative movement of the aa-tRNA with respect to EF-Tu. This relative rearrangement seems to facilitate codon recognition by the incoming aa-tRNA, and to provide the codon-anticodon recognition-dependent signal for the GTPase activity of EF-Tu. From these new findings we propose a mechanism that can explain the sequence of events during the decoding of mRNA on the ribosome.

RACK1 serves as a scaffold protein for a wide range of kinases and membrane-bound receptors. It is a WD-repeat family protein and is predicted to have a beta-propeller architecture with seven blades like a Gbeta protein. Mass spectrometry studies have identified its association with the small subunit of eukaryotic ribosomes and, most recently, it has been shown to regulate initiation by recruiting protein kinase C to the 40S subunit. Here we present the results of a cryo-EM study of the 80S ribosome that positively locate RACK1 on the head region of the 40S subunit, in the immediate vicinity of the mRNA exit channel. One face of RACK1 exposes the WD-repeats as a platform for interactions with kinases and receptors. Using this platform, RACK1 can recruit other proteins to the ribosome.

Cryo-EM density maps showing the 70S ribosome of E. coli in two different functional states related by a ratchet-like motion were analyzed using real-space refinement. Comparison of the two resulting atomic models shows that the ribosome changes from a compact structure to a looser one, coupled with the rearrangement of many of the proteins. Furthermore, in contrast to the unchanged inter-subunit bridges formed wholly by RNA, the bridges involving proteins undergo large conformational changes following the ratchet-like motion, suggesting an important role of ribosomal proteins in facilitating the dynamics of translation.

The receptor for activated C-kinase (RACK1) is a scaffold protein that is able to interact simultaneously with several signalling molecules. It binds to protein kinases and membrane-bound receptors in a regulated fashion. Interestingly, RACK1 is also a constituent of the eukaryotic ribosome, and a recent cryo-electron microscopy study localized it to the head region of the 40S subunit in the vicinity of the messenger RNA (mRNA) exit channel. RACK1 recruits activated protein kinase C to the ribosome, which leads to the stimulation of translation through the phosphorylation of initiation factor 6 and, potentially, of mRNAassociated proteins. RACK1 therefore links signal-transduction pathways directly to the ribosome, which allows translation to be regulated in response to cell stimuli. In addition, the fact that RACK1 associates with membrane-bound receptors indicates that it promotes the docking of ribosomes at sites where local translation is required, such as focal adhesions.

In translation, elongation factor Tu (EF-Tu) molecules deliver aminoacyl-tRNAs to the mRNA-programmed ribosome. The GTPase activity of EF-Tu is triggered by ribosome-induced conformational changes of the factor that play a pivotal role in the selection of the cognate aminoacyl-tRNAs. We present a 6.7-Å cryo-electron microscopy map of the aminoacyl-tRNA⅐EF-Tu⅐GDP⅐kirromycin-bound Escherichia coli ribosome, together with an atomic model of the complex obtained through molecular dynamics flexible fitting. The model reveals the conformational changes in the conserved GTPase switch regions of EF-Tu that trigger hydrolysis of GTP, along with key interactions, including those between the sarcin-ricin loop and the P loop of EF-Tu, and between the effector loop of EF-Tu and a conserved region of the 16S rRNA. Our data suggest that GTP hydrolysis on EF-Tu is controlled through a hydrophobic gate mechanism.decoding ͉ GTPase ͉ flexible fitting ͉ cryo-EM ͉ ternary complex

In the elongation cycle of translation, translocation is the process that advances the mRNA-tRNA moiety on the ribosome, to allow the next codon to move into the decoding center. New results obtained by cryoelectron microscopy, interpreted in the light of x-ray structures and kinetic data, allow us to develop a model of the molecular events during translocation.protein synthesis ͉ ribosome ͉ translation ͉ EF-G ͉ cryo-EM S ynthesis of proteins from their building blocks, the amino acids, is a fundamental process in the cells of all living organisms, be it animal, plant, or bacteria. The discovery that the macromolecular assembly that facilitates this process, the ribosome, is highly conserved in all essential parts has lent additional credence to the idea of the unity of all life at the molecular level.The ribosome is a very large (2.4 MDa in eubacteria) ribonucleicprotein complex composed of two distinct subunits, the small subunit (30S) charged with the task of decoding the genetic message carried by the messenger RNA (mRNA), the large subunit (50S) to the catalysis of peptide bond formation. Instrumental for these fundamental processes is the interaction of the ribosome with transfer RNA (tRNA), a small L-shaped molecule that embodies in its various forms the association of each amino acid with a threebase ''word'' of the genetic code, the codon. Translation is based on the mutual recognition, by partial Watson-Crick pairing, between the codon on the mRNA and the anticodon of the tRNA carrying the corresponding amino acid. In facilitating tRNA selection, decoding, and the stepwise formation of the polypeptide, ribosomal RNA (rRNA) acts as both a structural framework and a catalyst.Despite the success in the elucidation of ribosomal structure by x-ray crystallography, the detailed mechanism by which translation of mRNA code into peptide proceeds is still only scantly understood. One of the obstacles we face is that although the process is complex and dynamic, x-ray crystallography represents the molecule in a static form-packed in a crystal, moreover, whose very stability depends on intermolecular contacts that are largely nonphysiological. Of crucial importance for the understanding of the multistep translation process is the knowledge of how the ribosome interacts with its ligands, notably (apart from the most crucial ligands mRNA and tRNAs) the various factors catalyzing initiation, elongation, termination, and recycling. To date, with the exception of ribosomal complexes containing eubacterial release (RF1 or RF2) (1) or recycling (RRF) (2, 3) factors, there exists no x-ray structure of a factor-ribosome complex. The crystal structures of individual subunits complexed with initiation (4-6) and recycling (7) factors have also been solved; however, crystallographic data of elongation factors bound to the ribosome are currently still not available. Moreover, to date, despite many efforts, no atomic structure is available for a eukaryotic ribosome.Increasingly, within the past decade, cryo-electron microscopy (...

During the elongation cycle of protein biosynthesis, the speci®c amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl-tRNA, elongation factor Tu and GTP. As this ternary complex binds to the ribosome, the anticodon end of the tRNA reaches the decoding center in the 30S subunit. Here we present the cryoelectron microscopy (EM) study of an Escherichia coli 70S ribosome-bound ternary complex stalled with an antibiotic, kirromycin. In the cryo-EM map the anticodon arm of the tRNA presents a new conformation that appears to facilitate the initial codon±anticodon interaction. Furthermore, the elbow region of the tRNA is seen to contact the GTPase-associated center on the 50S subunit of the ribosome, suggesting an active role of the tRNA in the transmission of the signal prompting the GTP hydrolysis upon codon recognition.