Structure of the 30S ribosomal subunit

Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein-RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.     

A new understanding of the decoding principle on the ribosome

During protein synthesis, the ribosome accurately selects transfer RNAs (tRNAs) in accordance with the messenger RNA (mRNA) triplet in the decoding centre. tRNA selection is initiated by elongation factor Tu, which delivers tRNA to the aminoacyl tRNA-binding site (A site) and hydrolyses GTP upon establishing codon-anticodon interactions in the decoding centre. At the following proofreading step the ribosome re-examines the tRNA and rejects it if it does not match the A codon. It was suggested that universally conserved G530, A1492 and A1493 of 16S ribosomal RNA, critical for tRNA binding in the A site, actively monitor cognate tRNA, and that recognition of the correct codon-anticodon duplex induces an overall ribosome conformational change (domain closure). Here we propose an integrated mechanism for decoding based on six X-ray structures of the 70S ribosome determined at 3.1-3.4?? resolution, modelling cognate or near-cognate states of the decoding centre at the proofreading step. We show that the 30S subunit undergoes an identical domain closure upon binding of either cognate or near-cognate tRNA. This conformational change of the 30S subunit forms a decoding centre that constrains the mRNA in such a way that the first two nucleotides of the A codon are limited to form Watson-Crick base pairs. When U·G and G·U mismatches, generally considered to form wobble base pairs, are at the first or second codon-anticodon position, the decoding centre forces this pair to adopt the geometry close to that of a canonical C·G pair. This by itself, or with distortions in the codon-anticodon mini-helix and the anticodon loop, causes the near-cognate tRNA to dissociate from the ribosome.     

A ratchet-like inter-subunit reorganization of the ribosome during translocation

The ribosome is a macromolecular assembly that is responsible for protein biosynthesis following genetic instructions in all organisms. It is composed of two unequal subunits: the smaller subunit binds messenger RNA and the anticodon end of transfer RNAs, and helps to decode the mRNA; and the larger subunit interacts with the amino-acid-carrying end of tRNAs and catalyses the formation of the peptide bonds. After peptide-bond formation, elongation factor G (EF-G) binds to the ribosome, triggering the translocation of peptidyl-tRNA from its aminoacyl site to the peptidyl site, and movement of mRNA by one codon. Here we analyse three-dimensional cryo-electron microscopy maps of the Escherichia coli 70S ribosome in various functional states, and show that both EF-G binding and subsequent GTP hydrolysis lead to ratchet-like rotations of the small 30S subunit relative to the large 50S subunit. Furthermore, our finding indicates a two-step mechanism of translocation: first, relative rotation of the subunits and opening of the mRNA channel following binding of GTP to EF-G; and second, advance of the mRNA/(tRNA)2 complex in the direction of the rotation of the 30S subunit, following GTP hydrolysis.     

Structural basis for translation termination on the 70S ribosome

At termination of protein synthesis, type I release factors promote hydrolysis of the peptidyl-transfer RNA linkage in response to recognition of a stop codon. Here we describe the crystal structure of the Thermus thermophilus 70S ribosome in complex with the release factor RF1, tRNA and a messenger RNA containing a UAA stop codon, at 3.2 A resolution. The stop codon is recognized in a pocket formed by conserved elements of RF1, including its PxT recognition motif, and 16S ribosomal RNA. The codon and the 30S subunit A site undergo an induced fit that results in stabilization of a conformation of RF1 that promotes its interaction with the peptidyl transferase centre. Unexpectedly, the main-chain amide group of Gln 230 in the universally conserved GGQ motif of the factor is positioned to contribute directly to peptidyl-tRNA hydrolysis.     

Structure of the 30S translation initiation complex

Translation initiation, the rate-limiting step of the universal process of protein synthesis, proceeds through sequential, tightly regulated steps. In bacteria, the correct messenger RNA start site and the reading frame are selected when, with the help of initiation factors IF1, IF2 and IF3, the initiation codon is decoded in the peptidyl site of the 30S ribosomal subunit by the fMet-tRNA(fMet) anticodon. This yields a 30S initiation complex (30SIC) that is an intermediate in the formation of the 70S initiation complex (70SIC) that occurs on joining of the 50S ribosomal subunit to the 30SIC and release of the initiation factors. The localization of IF2 in the 30SIC has proved to be difficult so far using biochemical approaches, but could now be addressed using cryo-electron microscopy and advanced particle separation techniques on the basis of three-dimensional statistical analysis. Here we report the direct visualization of a 30SIC containing mRNA, fMet-tRNA(fMet) and initiation factors IF1 and GTP-bound IF2. We demonstrate that the fMet-tRNA(fMet) is held in a characteristic and precise position and conformation by two interactions that contribute to the formation of a stable complex: one involves the transfer RNA decoding stem which is buried in the 30S peptidyl site, and the other occurs between the carboxy-terminal domain of IF2 and the tRNA acceptor end. The structure provides insights into the mechanism of 70SIC assembly and rationalizes the rapid activation of GTP hydrolysis triggered on 30SIC-50S joining by showing that the GTP-binding domain of IF2 would directly face the GTPase-activated centre of the 50S subunit.     

A cryo-electron microscopic study of ribosome-bound termination factor RF2

Protein synthesis takes place on the ribosome, where genetic information carried by messenger RNA is translated into a sequence of amino acids. This process is terminated when a stop codon moves into the ribosomal decoding centre (DC) and is recognized by a class-1 release factor (RF). RFs have a conserved GGQ amino-acid motif, which is crucial for peptide release and is believed to interact directly with the peptidyl-transferase centre (PTC) of the 50S ribosomal subunit. Another conserved motif of RFs (SPF in RF2) has been proposed to interact directly with stop codons in the DC of the 30S subunit. The distance between the DC and PTC is approximately 73 A. However, in the X-ray structure of RF2, SPF and GGQ are only 23 A apart, indicating that they cannot be at DC and PTC simultaneously. Here we show that RF2 is in an open conformation when bound to the ribosome, allowing GGQ to reach the PTC while still allowing SPF-stop-codon interaction. The results indicate new interpretations of accuracy in termination, and have implications for how the presence of a stop codon in the DC is signalled to PTC.     

The structure of ribosomal protein S5 reveals sites of interaction with 16S rRNA.

Understanding the process whereby the ribosome translates the genetic code into protein molecules will ultimately require high-resolution structural information, and we report here the first crystal structure of a protein from the small ribosomal subunit. This protein, S5, has a molecular mass of 17,500 and is highly conserved in all lifeforms. The molecule contains two distinct alpha/beta domains that have structural similarities to several other proteins that are components of ribonucleoprotein complexes. Mutations in S5 result in several phenotypes which suggest that S5 may have a role in translational fidelity and translocation. These include ribosome ambiguity or ram, reversion from streptomycin dependence and resistance to spectinomycin. Also, a cold-sensitive, spectinomycin-resistant mutant of S5 has been identified which is defective in initiation. Here we show that these mutations map to two distinct regions of the molecule which seem to be sites of interaction with ribosomal RNA. A structure/function analysis of the molecule reveals discrepancies with current models of the 30S subunit.     

Structure of the Escherichia coli ribosomal termination complex with release factor 2

Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein-transfer-RNA bond hydrolysis. Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the 'SPF' loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.     

Structural basis for messenger RNA movement on the ribosome

Translation initiation is a major determinant of the overall expression level of a gene. The translation of functionally active protein requires the messenger RNA to be positioned on the ribosome such that the start/initiation codon will be read first and in the correct frame. Little is known about the molecular basis for the interaction of mRNA with the ribosome at different states of translation. Recent crystal structures of the ribosomal subunits, the empty 70S ribosome and the 70S ribosome containing functional ligands have provided information about the general organization of the ribosome and its functional centres. Here we compare the X-ray structures of eight ribosome complexes modelling the translation initiation, post-initiation and elongation states. In the initiation and post-initiation complexes, the presence of the Shine-Dalgarno (SD) duplex causes strong anchoring of the 5'-end of mRNA onto the platform of the 30S subunit, with numerous interactions between mRNA and the ribosome. Conversely, the 5' end of the 'elongator' mRNA lacking SD interactions is flexible, suggesting a different exit path for mRNA during elongation. After the initiation of translation, but while an SD interaction is still present, mRNA moves in the 3'-->5' direction with simultaneous clockwise rotation and lengthening of the SD duplex, bringing it into contact with ribosomal protein S2.     

Structure of a bacterial 30S ribosomal subunit at 5.5 A resolution.

The 30S ribosomal subunit binds messenger RNA and the anticodon stem-loop of transfer RNA during protein synthesis. A crystallographic analysis of the structure of the subunit from the bacterium Thermus thermophilus is presented. At a resolution of 5.5 A, the phosphate backbone of the ribosomal RNA is visible, as are the alpha-helices of the ribosomal proteins, enabling double-helical regions of RNA to be identified throughout the subunit, all seven of the small-subunit proteins of known crystal structure to be positioned in the electron density map, and the fold of the entire central domain of the small-subunit ribosomal RNA to be determined.     

Heterogeneous pathways and timing of factor departure during translation initiation

The initiation of translation establishes the reading frame for protein synthesis and is a key point of regulation. Initiation involves factor-driven assembly at a start codon of a messenger RNA of an elongation-competent 70S ribosomal particle (in bacteria) from separated 30S and 50S subunits and initiator transfer RNA. Here we establish in Escherichia coli, using direct single-molecule tracking, the timing of initiator tRNA, initiation factor 2 (IF2; encoded by infB) and 50S subunit joining during initiation. Our results show multiple pathways to initiation, with orders of arrival of tRNA and IF2 dependent on factor concentration and composition. IF2 accelerates 50S subunit joining and stabilizes the assembled 70S complex. Transition to elongation is gated by the departure of IF2 after GTP hydrolysis, allowing efficient arrival of elongator tRNAs to the second codon presented in the aminoacyl-tRNA binding site (A site). These experiments highlight the power of single-molecule approaches to delineate mechanisms in complex multicomponent systems.     

Concurrent nucleation of 16S folding and induced fit in 30S ribosome assembly

Rapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth. How the Escherichia coli 16S ribosomal RNA and the 20 proteins that make up the 30S ribosomal subunit can assemble correctly in a few minutes remains a challenging problem, partly because of the lack of real-time data on the earliest stages of assembly. By providing snapshots of individual RNA and protein interactions as they emerge in real time, here we show that 30S assembly nucleates concurrently from different points along the rRNA. Time-resolved hydroxyl radical footprinting was used to map changes in the structure of the rRNA within 20 milliseconds after the addition of total 30S proteins. Helical junctions in each domain fold within 100 ms. In contrast, interactions surrounding the decoding site and between the 5', the central and the 3' domains require 2-200 seconds to form. Unexpectedly, nucleotides contacted by the same protein are protected at different rates, indicating that initial RNA-protein encounter complexes refold during assembly. Although early steps in assembly are linked to intrinsically stable rRNA structure, later steps correspond to regions of induced fit between the proteins and the rRNA.     

The ribosome uses two active mechanisms to unwind messenger RNA during translation

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs.     

The joining of ribosomal subunits in eukaryotes requires eIF5B

Initiation of eukaryotic protein synthesis begins with the ribosome separated into its 40S and 60S subunits. The 40S subunit first binds eukaryotic initiation factor (eIF) 3 and an eIF2-GTP-initiator transfer RNA ternary complex. The resulting complex requires eIF1, eIF1A, eIF4A, eIF4B and eIF4F to bind to a messenger RNA and to scan to the initiation codon. eIF5 stimulates hydrolysis of eIF2-bound GTP and eIF2 is released from the 48S complex formed at the initiation codon before it is joined by a 60S subunit to form an active 80S ribosome. Here we show that hydrolysis of eIF2-bound GTP induced by eIF5 in 48S complexes is necessary but not sufficient for the subunits to join. A second factor termed eIF5B (relative molecular mass 175,000) is essential for this process. It is a homologue of the prokaryotic initiation factor IF2 (re and, like it, mediates joining of subunits and has a ribosome-dependent GTPase activity that is essential for its function.     

Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites

The elongation cycle of protein synthesis involves the delivery of aminoacyl-transfer RNAs to the aminoacyl-tRNA-binding site (A?site) of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A?site. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner. Despite the availability of structures of various EF-G-ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryoelectron microscopy analysis to resolve two previously unseen subpopulations within Thermus thermophilus EF-G-ribosome complexes at subnanometre resolution, one of them with a partly translocated tRNA. Comparison of these substates reveals that translocation of tRNA on the 30S subunit parallels the swivelling of the 30S head and is coupled to unratcheting of the 30S body. Because the tRNA maintains contact with the peptidyl-tRNA-binding site (P?site) on the 30S head and simultaneously establishes interaction with the exit site (E?site) on the 30S platform, a novel intra-subunit 'pe/E' hybrid state is formed. This state is stabilized by domain?IV of EF-G, which interacts with the swivelled 30S-head conformation. These findings provide direct structural and mechanistic insight into the 'missing link' in terms of tRNA intermediates involved in the universally conserved translocation process.     

SEC65 gene product is a subunit of the yeast signal recognition particle required for its integrity.

Protein targeting to the endoplasmic reticulum (ER) in mammalian cells is catalysed by the signal recognition particle (SRP), which consists of six protein subunits and an RNA subunit. Saccharomyces cerevisiae SRP is a 16S particle, of which only two subunits have been identified: a protein subunit, SRP54p, which is homologous to the mammalian SRP54 subunit, and an RNA subunit, scR1 (ref. 3). The sec65-1 mutant yeast cells are temperature-sensitive for growth and defective in the translocation of several secreted and membrane-bound proteins. The DNA sequence of the SEC65 gene suggests that its product is related to mammalian SRP19 subunit and may have a similar function. Here we show that SEC65p is a subunit of the S. cerevisiae SRP and that it is required for the stable association of another subunit, SRP54p, with SRP. Overexpression of SRP54p suppresses both growth and protein translocation defects in sec65-1 mutant cells.     

Antibiotic inhibition of group I ribozyme function

The discovery of catalytically active RNA has provided the basis for the evolutionary concept of an RNA world. It has been proposed that during evolution the functions of ancient catalytic RNA were modulated by low molecular weight effectors, related to antibiotics, present in the primordial soup. Antibiotics and RNA may have coevolved in the formation of the modern ribosome. Here we report that a set of aminoglycoside antibiotics, which are known to interact with the decoding region of the 16S ribosomal RNA of Escherichia coli, inhibit the second step of splicing of the T4 phage-derived td intron. Thus catalytic RNA seems to interact not only with a mononucleotide and an amino acid, but also with another class of biomolecules, the sugars. Splicing of other group I introns but not group II introns was inhibited. The similarity in affinity and specificity of these antibiotics for group I introns and rRNAs may result from recognition of evolutionarily conserved structures.     

A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting

The triplet-based genetic code requires that translating ribosomes maintain the reading frame of a messenger RNA faithfully to ensure correct protein synthesis. However, in programmed -1 ribosomal frameshifting, a specific subversion of frame maintenance takes place, wherein the ribosome is forced to shift one nucleotide backwards into an overlapping reading frame and to translate an entirely new sequence of amino acids. This process is indispensable in the replication of numerous viral pathogens, including HIV and the coronavirus associated with severe acute respiratory syndrome, and is also exploited in the expression of several cellular genes. Frameshifting is promoted by an mRNA signal composed of two essential elements: a heptanucleotide 'slippery' sequence and an adjacent mRNA secondary structure, most often an mRNA pseudoknot. How these components operate together to manipulate the ribosome is unknown. Here we describe the observation of a ribosome-mRNA pseudoknot complex that is stalled in the process of -1 frameshifting. Cryoelectron microscopic imaging of purified mammalian 80S ribosomes from rabbit reticulocytes paused at a coronavirus pseudoknot reveals an intermediate of the frameshifting process. From this it can be seen how the pseudoknot interacts with the ribosome to block the mRNA entrance channel, compromising the translocation process and leading to a spring-like deformation of the P-site transfer RNA. In addition, we identify movements of the likely eukaryotic ribosomal helicase and confirm a direct interaction between the translocase eEF2 and the P-site tRNA. Together, the structural changes provide a mechanical explanation of how the pseudoknot manipulates the ribosome into a different reading frame.     

Placement of protein and RNA structures into a 5 A-resolution map of the 50S ribosomal subunit.

We have calculated at 5.0 A resolution an electron-density map of the large 50S ribosomal subunit from the bacterium Haloarcula marismortui by using phases derived from four heavy-atom derivatives, intercrystal density averaging and density-modification procedures. More than 300 base pairs of A-form RNA duplex have been fitted into this map, as have regions of non-A-form duplex, single-stranded segments and tetraloops. The long rods of RNA crisscrossing the subunit arise from the stacking of short, separate double helices, not all of which are A-form, and in many places proteins crosslink two or more of these rods. The polypeptide exit channel was marked by tungsten cluster compounds bound in one heavy-atom-derivatized crystal. We have determined the structure of the translation-factor-binding centre by fitting the crystal structures of the ribosomal proteins L6, L11 and L14, the sarcin-ricin loop RNA, and the RNA sequence that binds L11 into the electron density. We can position either elongation factor G or elongation factor Tu complexed with an aminoacylated transfer RNA and GTP onto the factor-binding centre in a manner that is consistent with results from biochemical and electron microscopy studies.