TitleAuthorsAbstractInitiationThe elongation cycleDecodingPeptide-bond formationTranslocation: the formation of hybrid statesThe role of the E site in translocationThe role of EF-G in translocation relative to the 30S subunitTermination of translationRecognition of the stop codonCatalysis of peptide releaseThe role of RF3Recycling of ribosomes before reinitiationConclusionsReferencesFigure 1 Structure of the ribosome.Figure 2 Overview of bacterial translation.Figure 3 Decoding by the ribosome.Figure 4 Peptide-bond formation.Figure 5 EF-G catalysed translocation.Figure 6 Termination of translation by class I release factors.REVIEWSWhat recent ribosome structures haverevealed about the mechanism of translationT. Martin Schmeing1& V. Ramakrishnan1The high-resolution structures of ribosomal subunits published in 2000 have revolutionized the field of protein translation.They facilitated the determination and interp retation of functional complexes of the ribosome by crystallography andelectron microscopy. Knowledge of the precise positions of residues in the ribosome in various states has facilitatedincreasingly sophisticated biochemical and genetic experiments, as well as the use of new methods such as single-moleculekinetics. In this review, we discuss how the interaction between structural and functional studies over the last decade has ledto a deeper understanding of the complex mechanisms underlying translation.The ribosome is the large ribonucleoprotein particle thatsynthesizes proteins in all cells, using messenger RNA asthe template and aminoacyl-transfer RNAs as substrates.Ribosomes from bacteria consist of a large (50S) and a small(30S) subunit, which together compose the 2.5-megadalton 70S ribo-some; their eukaryotic counterparts are the 60S and 40S subunits andthe 80S ribosome. The 50S subunit consists of 23S RNA (,2,900nucleotides), 5S RNA (,120 nucleotides) and about 30 proteins;the 30S subunit consists of 16S RNA (,1,500 nucleotides) and about20 proteins. In addition, several protein factors act on the ribosome atvarious stages of translation. In this review, we focus mainly onstructural and mechanistic insights into bacterial translationobtained in the last few years. A previous review deals more exten-sively with earlier work1.The essentially complete atomic structures of an archaeal 50S subunitfrom Haloarcula marismortui2and a bacterial 30S subunit fromThermus thermophilus3published in 2000 were the basis for the phasingand/or molecular interpretation of every subsequent structure of theribosome or its subunits. Such structures include low-resolution struc-tures of the 70S ribosome by crystallography4or cryoelectron micro-scopy (cryoEM)5, the structure of a bacterial 50S subunit6, and morerecent high-resolution structures of the 70S ribosome7,8.Finally,mobileelements of the 50S subunit such as the L1 or L7/L12 stalks that arepartly or completely disordered in most high-resolution structures oftheribosomeorthe50Ssubunithavebeensolvedinisolation9,10.The basic architecture of the ribosome is shown in Fig. 1. Theinterface between the two subunits consists mainly of RNA. ThemRNA binds in a cleft between the ‘head’ and ‘body’ of the 30Ssubunit, where its codons interact with the anticodons of tRNA.There are three binding sites for tRNA: the A site that binds theincoming aminoacyl-tRNA, the P site that holds the peptidyl-tRNAattached to the nascent polypeptide chain, and the E (exit) site towhich the deacylated P-site tRNA moves after peptide-bond forma-tion before its ejection from the ribosome. In the 50S subunit, the 39ends of P- and A-site tRNAs are in close proximity in the peptidyl-transferase centre (PTC), whereas the 39 end of the E-site tRNA is,50 A˚away from the PTC.InitiationBacterial translation can be roughly divided into three main stages,initiation, elongation and termination (Fig. 2; a movie of the processcan be seen at http://www.mrc-lmb.cam.ac.uk/ribo/homepage/movies/translation_bacterial.mov). Initiation requires the ribosometo position the initiator fMet-tRNAfMetover the start codon ofmRNA in the P site. In bacteria, the ribosome is positioned in thevicinity of the start codon by base pairing between the 39 end of 16SRNA and an approximately complementary sequence just upstreamof the mRNA start codon, called the Shine–Dalgarno sequence. Theprecise positioning of the start codon in the P site requires the bind-ing of a special initiator fMet-tRNAfMetand three initiation factors,IF1–3. However, exactly how the correct tRNA is selected remainsunclear, as are the roles of the various factors.A probable first step in initiation is the binding of IF3 to the 30S thathas been split from the 50S by ribosome recycling factor RRF andelongation factor G (EF-G) after translational termination (see Fig. 2and the termination section later). This binding stimulates release ofthe mRNA and deacylated tRNA, leftover from the previous round oftranslation, from the 30S and prevents the large subunit from re-associating11,12. The binding of the 30S–IF3 complex to mRNA, IF1,IF2 and initiator tRNA results in the 30S initiation complex (30S-IC).IF2, a GTPase, promotes subunit joining to form the 70S initiationcomplex (70S-IC), which is accompanied by IF3 release13–15. AfterGTP hydrolysis and phosphate release from IF2 (refs 16, 17), fMet-tRNAfMetmoves into the PTC, readying the ribosome for elongation.The mechanism of initiation is still unclear, owing to a paucity ofstructural data. There has been little progress towards high-resolutionstructures of initiation complexes since the structure of IF1 bound to a30S subunit18. However, recent cryoEM studies have visualized both30S and 70S initiation complexes. In a 30S-IC (ref. 19), which un-fortunately did not contain IF3, IF2 stretches across the subunit inter-face of the 30S, contacting the acceptor end of fMet-tRNAfMetwith itscarboxy terminus. The anticodon stem and elbow are shifted towardsthe E site, resulting in a ‘30S P/I state’. IF1 is visible in the A site, but doesnot contact IF2. After subunit joining, the G domain of IF2 interactswith the GTPase centre of the large subunit20. It maintains its contactswith fMet-tRNAfMet, which has shifted up out of plane from the 30S P/Istatetoa70SP/Istate,andseemstomakeadirectcontactwithIF1inthe70S-IC. The 30S subunit is rotated relative to the 50S by ,4u anticlock-wise, similar to the ratcheting seen during translocation21.In the structure of