Vol 461j29 October 2009jdoi 10 1038 nature08403 REVIEWS What recent ribosome structures have revealed about the mechanism of translation T Martin Schmeing1 V Ramakrishnan1 The high resolution structures of ribosomal subunits published in 2000 have revolutionized the field of protein translation They facilitated the determination and interpretation of functional complexes of the ribosome by crystallography and electron microscopy Knowledge of the precise positions of residues in the ribosome in various states has facilitated increasingly sophisticated biochemical and genetic experiments as well as the use of new methods such as single molecule kinetics In this review we discuss how the interaction between structural and functional studies over the last decade has led to a deeper understanding of the complex mechanisms underlying translation he ribosome is the large ribonucleoprotein particle that synthesizes proteins in all cells using messenger RNA as the 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 ribosome their eukaryotic counterparts are the 60S and 40S subunits and the 80S ribosome The 50S subunit consists of 23S RNA 2 900 nucleotides 5S RNA 120 nucleotides and about 30 proteins the 30S subunit consists of 16S RNA 1 500 nucleotides and about 20 proteins In addition several protein factors act on the ribosome at various stages of translation In this review we focus mainly on structural and mechanistic insights into bacterial translation obtained in the last few years A previous review deals more extensively with earlier work1 The essentially complete atomic structures of an archaeal 50S subunit from Haloarcula marismortui2 and a bacterial 30S subunit from Thermus thermophilus3 published in 2000 were the basis for the phasing and or molecular interpretation of every subsequent structure of the ribosome or its subunits Such structures include low resolution structures of the 70S ribosome by crystallography4 or cryoelectron microscopy cryoEM 5 the structure of a bacterial 50S subunit6 and more recent high resolution structures of the 70S ribosome7 8 Finally mobile elements of the 50S subunit such as the L1 or L7 L12 stalks that are partly or completely disordered in most high resolution structures of the ribosome or the 50S subunit have been solved in isolation9 10 The basic architecture of the ribosome is shown in Fig 1 The interface between the two subunits consists mainly of RNA The mRNA binds in a cleft between the head and body of the 30S subunit where its codons interact with the anticodons of tRNA There are three binding sites for tRNA the A site that binds the incoming aminoacyl tRNA the P site that holds the peptidyl tRNA attached to the nascent polypeptide chain and the E exit site to which the deacylated P site tRNA moves after peptide bond formation before its ejection from the ribosome In the 50S subunit the 39 ends of P and A site tRNAs are in close proximity in the peptidyltransferase centre PTC whereas the 39 end of the E site tRNA is 50 A away from the PTC T Initiation Bacterial translation can be roughly divided into three main stages initiation elongation and termination Fig 2 a movie of the process 1 can be seen at http www mrc lmb cam ac uk ribo homepage movies translation bacterial mov Initiation requires the ribosome to position the initiator fMet tRNAfMet over the start codon of mRNA in the P site In bacteria the ribosome is positioned in the vicinity of the start codon by base pairing between the 39 end of 16S RNA and an approximately complementary sequence just upstream of the mRNA start codon called the Shine Dalgarno sequence The precise positioning of the start codon in the P site requires the binding of a special initiator fMet tRNAfMet and three initiation factors IF1 3 However exactly how the correct tRNA is selected remains unclear as are the roles of the various factors A probable first step in initiation is the binding of IF3 to the 30S that has been split from the 50S by ribosome recycling factor RRF and elongation factor G EF G after translational termination see Fig 2 and the termination section later This binding stimulates release of the mRNA and deacylated tRNA leftover from the previous round of translation from the 30S and prevents the large subunit from reassociating11 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 initiation complex 70S IC which is accompanied by IF3 release13 15 After GTP hydrolysis and phosphate release from IF2 refs 16 17 fMettRNAfMet moves into the PTC readying the ribosome for elongation The mechanism of initiation is still unclear owing to a paucity of structural data There has been little progress towards high resolution structures of initiation complexes since the structure of IF1 bound to a 30S subunit18 However recent cryoEM studies have visualized both 30S and 70S initiation complexes In a 30S IC ref 19 which unfortunately did not contain IF3 IF2 stretches across the subunit interface of the 30S contacting the acceptor end of fMet tRNAfMet with its carboxy terminus The anticodon stem and elbow are shifted towards the E site resulting in a 30S P I state IF1 is visible in the A site but does not contact IF2 After subunit joining the G domain of IF2 interacts with the GTPase centre of the large subunit20 It maintains its contacts with fMet tRNAfMet which has shifted up out of plane from the 30S P I state to a 70S P I state and seems to make a direct contact with IF1 in the 70S IC The 30S subunit is rotated relative to the 50S by 4u anticlockwise similar to the ratcheting seen during translocation21 In the structure of 70S mRNA fMet tRNAfMet IF2 GDPCP22 IF2 is still bound to the GTPase centre but has lost contact with fMet tRNAfMet now in the PTC in the canonical P P state The MRC Laboratory of Molecular Biology Cambridge CB2 0QH UK 1234 2009 Macmillan Publishers Limited All rights reserved REVIEWS NATUREjVol 461j29 October 2009 a P tRNA 5S A tRNA 50S L1 L7 L12 E tRNA Body 5 Head 30S mRNA 3 b c CP E tRNA Head L1 P tRNA E tRNA A tRNA Beak L7 L12 P tRNA 3 mRNA GTPase factor binding site A tRNA DC PTC Body 30S 50S Spur Figure 1 Structure of the ribosome a Top view of the 70S ribosome with mRNA and A P and E site tRNAs b c Exploded view of the 30S subunit b and 50S