An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA

The large ribosomal subunit catalyses the reaction between the alpha-amino group of the aminoacyl-tRNA bound to the A site and the ester carbon of the peptidyl-tRNA bound to the P site, while preventing the nucleophilic attack of water on the ester, which would lead to unprogrammed deacylation of the peptidyl-tRNA. Here we describe three new structures of the large ribosomal subunit of Haloarcula marismortui (Hma) complexed with peptidyl transferase substrate analogues that reveal an induced-fit mechanism in which substrates and active-site residues reposition to allow the peptidyl transferase reaction. Proper binding of an aminoacyl-tRNA analogue to the A site induces specific movements of 23S rRNA nucleotides 2618-2620 (Escherichia coli numbering 2583-2585) and 2541(2506), thereby reorienting the ester group of the peptidyl-tRNA and making it accessible for attack. In the absence of the appropriate A-site substrate, the peptidyl transferase centre positions the ester link of the peptidyl-tRNA in a conformation that precludes the catalysed nucleophilic attack by water. Protein release factors may also function, in part, by inducing an active-site rearrangement similar to that produced by the A-site aminoacyl-tRNA, allowing the carbonyl group and water to be positioned for 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.     

Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria.

Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavity and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg+2 ions for the binding of some drugs. This structural analysis should facilitate rational drug design.     

肽酰转移酶催化肽键形成机理的理论计算研究

蛋白质合成过程关键的一步,由肽酰转移酶参与的肽键形成机理是一个基础的被长期讨论的生物分子学问题．早在三十几年前,就已经知道肽酰转移酶对肽键的合成起至关重要的作用．并且也知道肽酰转移酶是一个大的核蛋白,即包括很多蛋白质和RNA的大分子．但是一直以来科学家都认为,这个反应机理应该和丝氨酸水解酶水解蛋白质一样,是一个基于蛋白质催化的过程．     

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.     

Different substrate-dependent transition states in the active site of the ribosome

The active site of the ribosome, the peptidyl transferase centre, catalyses two reactions, namely, peptide bond formation between peptidyl-tRNA and aminoacyl-tRNA as well as the release-factor-dependent hydrolysis of peptidyl-tRNA. Unlike peptide bond formation, peptide release is strongly impaired by mutations of nucleotides within the active site, in particular by base exchanges at position A2602 (refs 1, 2). The 2'-OH group of A76 of the peptidyl-tRNA substrate seems to have a key role in peptide release. According to computational analysis, the 2'-OH may take part in a concerted 'proton shuttle' by which the leaving group is protonated, in analogy to similar current models of peptide bond formation. Here we report kinetic solvent isotope effects and proton inventories (reaction rates measured in buffers with increasing content of deuterated water, D(2)O) of the two reactions catalysed by the active site of the Escherichia coli ribosome. The transition state of the release factor 2 (RF2)-dependent hydrolysis reaction is characterized by the rate-limiting formation of a single strong hydrogen bond. This finding argues against a concerted proton shuttle in the transition state of the hydrolysis reaction. In comparison, the proton inventory for peptide bond formation indicates the rate-limiting formation of three hydrogen bonds with about equal contributions, consistent with a concerted eight-membered proton shuttle in the transition state. Thus, the ribosome supports different rate-limiting transition states for the two reactions that take place in the peptidyl transferase centre.     

蛋白水解酶转肽使猪胰岛素转成人胰岛素

利用赖氨酸专一性的蛋白水解酶进行了酶促人胰岛素半合成反应，猪胰岛素经该酶切去Ｂ链羧基端丙氨酸形成去丙氨酸胰岛素素ＤＡＩ。进一步在水／有机溶剂介质中该酶的转肽反应，使Ｂ链生成苏氨酸叔丁酯羧基末端，水解去酯后产生人胰岛素。研究了转肽反应的最佳条件。转肽反应生成的人胰岛素具有较强的生物活性。     

Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics

The 30S ribosomal subunit has two primary functions in protein synthesis. It discriminates against aminoacyl transfer RNAs that do not match the codon of messenger RNA, thereby ensuring accuracy in translation of the genetic message in a process called decoding. Also, it works with the 50S subunit to move the tRNAs and associated mRNA by precisely one codon, in a process called translocation. Here we describe the functional implications of the high-resolution 30S crystal structure presented in the accompanying paper, and infer details of the interactions between the 30S subunit and its tRNA and mRNA ligands. We also describe the crystal structure of the 30S subunit complexed with the antibiotics paromomycin, streptomycin and spectinomycin, which interfere with decoding and translocation. This work reveals the structural basis for the action of these antibiotics, and leads to a model for the role of the universally conserved 16S RNA residues A1492 and A1493 in the decoding process.     

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.     

Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA

The elongation factors EF-Tu and EF-G interact with ribosomes during protein synthesis: EF-Tu presents incoming aminoacyl transfer RNA to the programmed ribosome as an EF-Tu-GTP-tRNA ternary complex and EF-G promotes translocation of peptidyl-tRNA and its associated messenger RNA from the A to the P site after peptidyl transfer. Both events are accompanied by ribosome-dependent GTP hydrolysis. Here we use chemical probes to investigate the possible interaction of these factors with ribosomal RNA in E. coli ribosomes. We observe EF-G-dependent footprints in vitro and in vivo around position 1,067 in domain II of 23S rRNA, and in the loop around position 2,660 in domain VI.EF-Tu gives an overlapping footprint in vitro at positions 2,655 and 2,661, but shows no effect at position 1,067. The 1,067 region is the site of interaction of the antibiotic thiostrepton, which prevents formation of the EF-G-GTP-ribosome complex and is a site for interaction with the GTPase-related protein L11 (ref. 3). The universally conserved loop in the 2,660 region is the site of attack by the RNA-directed cytotoxins alpha-sarcin and ricin, whose effects abolish translation and include the loss of elongation factor-dependent functions in eukaryotic ribosomes.     

Peptide bond formation destabilizes Shine-Dalgarno interaction on the ribosome

The ribosome is a molecular machine that translates the genetic code contained in the messenger RNA into an amino acid sequence through repetitive cycles of transfer RNA selection, peptide bond formation and translocation. Here we demonstrate an optical tweezer assay to measure the rupture force between a single ribosome complex and mRNA. The rupture force was compared between ribosome complexes assembled on an mRNA with and without a strong Shine-Dalgarno (SD) sequence-a sequence found just upstream of the coding region of bacterial mRNAs, involved in translation initiation. The removal of the SD sequence significantly reduced the rupture force in complexes carrying an aminoacyl tRNA, Phe-tRNA(Phe), in the A site, indicating that the SD interactions contribute significantly to the stability of the ribosomal complex on the mRNA before peptide bond formation. In contrast, the presence of a peptidyl tRNA analogue, N-acetyl-Phe-tRNA(Phe), in the A site, which mimicked the post-peptidyl transfer state, weakened the rupture force as compared to the complex with Phe-tRNA(Phe), and the resultant force was the same for both the SD-containing and SD-deficient mRNAs. These results suggest that formation of the first peptide bond destabilizes the SD interaction, resulting in the weakening of the force with which the ribosome grips an mRNA. This might be an important requirement to facilitate movement of the ribosome along mRNA during the first translocation step.     

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.     

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.     

Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit

The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments. Along this pathway, multiple pre-ribosomal particles are generated, which transiently associate with numerous non-ribosomal factors before mature 60S and 40S subunits are formed. However, most mechanistic details of ribosome biogenesis are still unknown. Here we identify a maturation step of the yeast pre-40S subunit that is regulated by the protein kinase Hrr25 and involves ribosomal protein Rps3. A high salt concentration releases Rps3 from isolated pre-40S particles but not from mature 40S subunits. Electron microscopy indicates that pre-40S particles lack a structural landmark present in mature 40S subunits, the 'beak'. The beak is formed by the protrusion of 18S ribosomal RNA helix 33, which is in close vicinity to Rps3. Two protein kinases Hrr25 and Rio2 are associated with pre-40S particles. Hrr25 phosphorylates Rps3 and the 40S synthesis factor Enp1. Phosphorylated Rsp3 and Enp1 readily dissociate from the pre-ribosome, whereas subsequent dephosphorylation induces formation of the beak structure and salt-resistant integration of Rps3 into the 40S subunit. In vivo depletion of Hrr25 inhibits growth and leads to the accumulation of immature 40S subunits that contain unstably bound Rps3. We conclude that the kinase activity of Hrr25 regulates the maturation of 40S ribosomal subunits.     

The RNA required in the first step of chlorophyll biosynthesis is a chloroplast glutamate tRNA

A molecule of chlorophyll is synthesized from eight molecules of delta-aminolevulinate (DALA), the universal precursor of porphyrins. The light-regulated conversion of glutamate to delta-aminolevulinate in the stroma of greening plastids involves the reduction of glutamate to glutamate-1-semialdehyde and its subsequent transamination. The components performing this conversion have been isolated from barley and Chlamydomonas and separated into three fractions by serial affinity chromatography on Blue Sepharose and haem- or chlorophyllin-Sepharose. The complete reaction can be performed in vitro in a reconstituted assay by combining all three fractions. An RNA is the essential component of the chlorophyllin-Sepharose-bound fraction. By nucleotide sequence analysis, we have now identified this RNA as a chloroplast glutamate acceptor RNA. Glutamate attached by an aminoacyl bond to the 3'-terminal adenosine of this RNA is a substrate for the enzyme(s) which perform the subsequent reactions. This reaction represents a novel role for transfer RNA: participation in the metabolic conversion of its cognate amino acid into another metabolite of low relative molecular mass which subsequently is not used in peptide bond synthesis.     

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.     

Cleavage of pre-tRNAs by the splicing endonuclease requires a composite active site

Splicing is required for the removal of introns from a subset of transfer RNAs in all eukaryotic organisms. The first step of splicing, intron recognition and cleavage, is performed by the tRNA-splicing endonuclease, a tetrameric enzyme composed of the protein subunits Sen54, Sen2, Sen34 and Sen15. It has previously been demonstrated that the active sites for cleavage at the 5' and 3' splice sites of precursor tRNA are contained within Sen2 and Sen34, respectively. A recent structure of an archaeal endonuclease complexed with a bulge-helix-bulge RNA has led to the unexpected hypothesis that catalysis requires a critical 'cation-pi sandwich' composed of two arginine residues that serve to position the RNA substrate within the active site. This motif is derived from a cross-subunit interaction between the two catalytic subunits. Here we test the role of this interaction within the eukaryotic endonuclease and show that catalysis at the 5' splice site requires the conserved cation-pi sandwich derived from the Sen34 subunit in addition to the catalytic triad of Sen2. The catalysis of pre-tRNA by the eukaryotic tRNA-splicing endonuclease therefore requires a previously unrecognized composite active site.     

Natural products as inspiration for the development of asymmetric catalysis

Biologically active natural products often contain particularly challenging structural features and functionalities in terms of synthesis. Perhaps the greatest difficulties are those caused by issues of stereochemistry. A useful strategy for synthesizing such molecules is to devise methods of bond formation that provide opportunities for using enantioselective catalysis. In using this tactic, the desire for a particular target structure ultimately drives the development of catalytic methods. New enantioselective catalytic methods contribute to a greater fundamental understanding of how bonds can be constructed and lead to valuable synthetic technologies that are useful for a variety of applications.