ABSTRACT
In this Review, we explore natural product antibiotics that do more than simply inhibit an active site of an essential enzyme. We review these compounds to provide inspiration for the design of much-needed new antibacterial agents, and examine the complex mechanisms that have evolved to effectively target bacteria, including covalent binders, inhibitors of resistance, compounds that utilize self-promoted entry, those that evade resistance, prodrugs, target corrupters, inhibitors of 'undruggable' targets, compounds that form supramolecular complexes, and selective membrane-acting agents. These are exemplified by ß-lactams that bind covalently to inhibit transpeptidases and ß-lactamases, siderophore chimeras that hijack import mechanisms to smuggle antibiotics into the cell, compounds that are activated by bacterial enzymes to produce reactive molecules, and antibiotics such as aminoglycosides that corrupt, rather than merely inhibit, their targets. Some of these mechanisms are highly sophisticated, such as the preformed ß-strands of darobactins that target the undruggable ß-barrel chaperone BamA, or teixobactin, which binds to a precursor of peptidoglycan and then forms a supramolecular structure that damages the membrane, impeding the emergence of resistance. Many of the compounds exhibit more than one notable feature, such as resistance evasion and target corruption. Understanding the surprising complexity of the best antimicrobial compounds provides a roadmap for developing novel compounds to address the antimicrobial resistance crisis by mining for new natural products and inspiring us to design similarly sophisticated antibiotics.
Subject(s)
Anti-Bacterial Agents , Bacteria , Biological Products , Animals , Humans , Aminoglycosides/pharmacology , Aminoglycosides/chemistry , Aminoglycosides/metabolism , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Bacteria/drug effects , Bacteria/enzymology , Bacteria/metabolism , beta Lactam Antibiotics/chemistry , beta Lactam Antibiotics/pharmacology , beta-Lactamase Inhibitors/chemistry , beta-Lactamase Inhibitors/pharmacology , Biological Products/chemistry , Biological Products/pharmacology , Biological Products/metabolism , Drug Design , Drug Resistance, Bacterial/drug effects , Peptidyl Transferases/antagonists & inhibitors , Prodrugs/pharmacology , Prodrugs/chemistry , Prodrugs/metabolism , Siderophores/metabolism , Siderophores/chemistry , Siderophores/pharmacologyABSTRACT
Ribosome frameshifting during translation of bacterial dnaX can proceed via different routes, generating a variety of distinct polypeptides. Using kinetic experiments, we show that -1 frameshifting predominantly occurs during translocation of two tRNAs bound to the slippery sequence codons. This pathway depends on a stem-loop mRNA structure downstream of the slippery sequence and operates when aminoacyl-tRNAs are abundant. However, when aminoacyl-tRNAs are in short supply, the ribosome switches to an alternative frameshifting pathway that is independent of a stem-loop. Ribosome stalling at a vacant 0-frame A-site codon results in slippage of the P-site peptidyl-tRNA, allowing for -1-frame decoding. When the -1-frame aminoacyl-tRNA is lacking, the ribosomes switch into -2 frame. Quantitative mass spectrometry shows that the -2-frame product is synthesized in vivo. We suggest that switching between frameshifting routes may enrich gene expression at conditions of aminoacyl-tRNA limitation.
Subject(s)
Bacterial Proteins/biosynthesis , DNA Polymerase III/biosynthesis , Escherichia coli/enzymology , Frameshifting, Ribosomal , RNA, Bacterial/metabolism , RNA, Messenger/metabolism , RNA, Transfer, Amino Acyl/metabolism , Bacterial Proteins/genetics , DNA Polymerase III/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Kinetics , Mutation , Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Transfer, Amino Acyl/chemistry , RNA, Transfer, Amino Acyl/genetics , Spectrometry, Mass, Electrospray Ionization , Structure-Activity Relationship , Tandem Mass SpectrometryABSTRACT
Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Peptide Elongation Factors/metabolism , Peptides/metabolism , Ribosomes/metabolism , Binding Sites , Cryoelectron Microscopy , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/ultrastructure , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Nucleic Acid Conformation , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/genetics , Peptide Elongation Factors/ultrastructure , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/metabolism , Peptides/chemistry , Protein Binding , Protein Biosynthesis , Protein Conformation , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer/chemistry , RNA, Transfer/genetics , RNA, Transfer/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Ribosomes/chemistry , Ribosomes/ultrastructure , Structure-Activity Relationship , Eukaryotic Translation Initiation Factor 5AABSTRACT
During canonical translation, the ribosome moves along an mRNA from the start to the stop codon in exact steps of one codon at a time. The collinearity of the mRNA and the protein sequence is essential for the quality of the cellular proteome. Spontaneous errors in decoding or translocation are rare and result in a deficient protein. However, dedicated recoding signals in the mRNA can reprogram the ribosome to read the message in alternative ways. This review summarizes the recent advances in understanding the mechanisms of three types of recoding events: stop-codon readthrough, -1 ribosome frameshifting and translational bypassing. Recoding events provide insights into alternative modes of ribosome dynamics that are potentially applicable to other non-canonical modes of prokaryotic and eukaryotic translation.
Subject(s)
Protein Biosynthesis , Codon, Terminator , Frameshifting, Ribosomal , Ribosomes/metabolismABSTRACT
Assessment of the fidelity of gene expression is crucial to understand cell homeostasis. Here we present a highly sensitive method for the systematic Quantification of Rare Amino acid Substitutions (QRAS) using absolute quantification by targeted mass spectrometry after chromatographic enrichment of peptides with missense amino acid substitutions. By analyzing incorporation of near- and non-cognate amino acids in a model protein EF-Tu, we show that most of missense errors are too rare to detect by conventional methods, such as DDA, and are estimated to be between <10-7-10-5 by QRAS. We also observe error hotspots of up to 10-3 for some types of mismatches, including the G-U mismatch. The error frequency depends on the expression level of EF-Tu and, surprisingly, the amino acid position in the protein. QRAS is not restricted to any particular miscoding event, organism, strain or model protein and is a reliable tool to analyze very rare proteogenomic events.
Subject(s)
Escherichia coli Proteins/genetics , Gene Expression/genetics , Mutation, Missense/genetics , Peptide Elongation Factor Tu/genetics , Amino Acids , Escherichia coli/genetics , Homeostasis/geneticsABSTRACT
The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A- and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally. Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome.
Subject(s)
Protein Biosynthesis , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Cold Shock Proteins and Peptides/biosynthesis , Cold Shock Proteins and Peptides/chemistry , Escherichia coli , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/chemistry , Kinetics , Ribosomal Proteins/physiology , Ribosomes/physiologyABSTRACT
A majority of cellular functions are carried out by macromolecular complexes. A host of biochemical and spectroscopic methods exists to characterize especially protein/protein complexes, however there has been a lack of a universal method to determine protein stoichiometries. Peptide-based MS, especially as a complementary method to the MS analysis of intact protein complexes, has now been developed to a point where it can be employed to assay protein stoichiometries in a routine manner. While the experimental demands are still significant, peptide-based MS has been successfully applied to analyze stoichiometries for a variety of protein complexes from very different biological backgrounds. In this review, we discuss the requirements especially for targeted MS acquisition strategies to be used in this context, with a special focus on the interconnected experimental aspects of sample preparation, protein digestion, and peptide stability. In addition, different strategies for the introduction of quantitative peptide standards and their suitability for different scenarios are compared.
Subject(s)
Macromolecular Substances , Mass Spectrometry/methods , Peptides , Macromolecular Substances/analysis , Macromolecular Substances/chemistry , Peptides/analysis , Peptides/chemistry , ProteomicsABSTRACT
The peptide bond formation with the amino acid proline (Pro) on the ribosome is slow, resulting in translational stalling when several Pro have to be incorporated into the peptide. Stalling at poly-Pro motifs is alleviated by the elongation factor P (EF-P). Here we investigate why Pro is a poor substrate and how EF-P catalyzes the reaction. Linear free energy relationships of the reaction on the ribosome and in solution using 12 different Pro analogues suggest that the positioning of Pro-tRNA in the peptidyl transferase center is the major determinant for the slow reaction. With any Pro analogue tested, EF-P decreases the activation energy of the reaction by an almost uniform value of 2.5 kcal/mol. The main source of catalysis is the favorable entropy change brought about by EF-P. Thus, EF-P acts by entropic steering of Pro-tRNA toward a catalytically productive orientation in the peptidyl transferase center of the ribosome.
Subject(s)
Entropy , Peptide Elongation Factors/chemistry , Proline/chemistry , Ribosomes/chemistry , Catalytic DomainABSTRACT
The speed and accuracy of protein synthesis are fundamental parameters for understanding the fitness of living cells, the quality control of translation, and the evolution of ribosomes. In this study, we analyse the speed and accuracy of the decoding step under conditions reproducing the high speed of translation in vivo. We show that error frequency is close to 10⻳, consistent with the values measured in vivo. Selectivity is predominantly due to the differences in k(cat) values for cognate and near-cognate reactions, whereas the intrinsic affinity differences are not used for tRNA discrimination. Thus, the ribosome seems to be optimized towards high speed of translation at the cost of fidelity. Competition with near- and non-cognate ternary complexes reduces the rate of GTP hydrolysis in the cognate ternary complex, but does not appreciably affect the rate-limiting tRNA accommodation step. The GTP hydrolysis step is crucial for the optimization of both the speed and accuracy, which explains the necessity for the trade-off between the two fundamental parameters of translation.
Subject(s)
Codon/genetics , Guanosine Triphosphate/metabolism , Peptide Chain Elongation, Translational , Protein Biosynthesis , RNA, Transfer, Amino Acid-Specific , RNA, Transfer, Amino Acyl , Ribosomes/metabolism , Escherichia coli , KineticsABSTRACT
Aminoglycoside antibiotics target the ribosome and induce mistranslation, yet which translation errors induce bacterial cell death is unclear. The analysis of cellular proteins by quantitative mass spectrometry shows that bactericidal aminoglycosides induce not only single translation errors, but also clusters of errors in full-length proteins in vivo with as many as four amino acid substitutions in a row. The downstream errors in a cluster are up to 10,000-fold more frequent than the first error and independent of the intracellular aminoglycoside concentration. The prevalence, length, and composition of error clusters depends not only on the misreading propensity of a given aminoglycoside, but also on its ability to inhibit ribosome translocation along the mRNA. Error clusters constitute a distinct class of misreading events in vivo that may provide the predominant source of proteotoxic stress at low aminoglycoside concentration, which is particularly important for the autocatalytic uptake of the drugs.
Subject(s)
Aminoglycosides/pharmacology , Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Peptide Elongation Factor Tu/metabolism , Protein Biosynthesis/genetics , Proteome/genetics , Ribosomes/metabolism , Stress, Physiological/drug effects , Escherichia coli/genetics , Escherichia coli/metabolism , Mass Spectrometry , Mutation, Missense , Nebramycin/analogs & derivatives , Nebramycin/pharmacology , Peptide Elongation Factor Tu/genetics , Peptides/genetics , Peptides/metabolism , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , Proteome/drug effects , Proteome/metabolism , Proteomics , Recombinant Proteins , Ribosomes/drug effects , Streptomycin/pharmacology , Stress, Physiological/geneticsABSTRACT
The ribosome has an active site comprised of RNA that catalyzes peptide bond formation. To understand how RNA promotes this reaction requires a detailed understanding of the chemical transition state. Here, we report the Brønsted coefficient of the alpha-amino nucleophile with a series of puromycin derivatives. Both 50S subunit- and 70S ribosome-catalyzed reactions displayed linear free-energy relationships with slopes close to zero under conditions where chemistry is rate limiting. These results indicate that, at the transition state, the nucleophile is neutral in the ribosome-catalyzed reaction, in contrast to the substantial positive charge reported for typical uncatalyzed aminolysis reactions. This suggests that the ribosomal transition state involves deprotonation to a degree commensurate with nitrogen-carbon bond formation. Such a transition state is significantly different from that of uncatalyzed aminolysis reactions in solution.
Subject(s)
Amines/chemistry , Ribosomal Proteins/chemistryABSTRACT
Release factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET and biochemical assays, we show that ribosome termination complexes that carry two factors, RF1-RF3 or RF2-RF3, are dynamic and fluctuate between non-rotated and rotated states, whereas each factor alone has its distinct signature on ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. In the absence of RF1, RF3 is stalled on ribosomes if GTP hydrolysis is blocked. Our data suggest how the assembly of the ribosome-RF1-RF3-GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.
Subject(s)
Escherichia coli Proteins/genetics , Escherichia coli/genetics , Peptide Chain Termination, Translational , Peptide Termination Factors/genetics , RNA, Transfer, Amino Acyl/genetics , Ribosomes/genetics , Carbocyanines/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Guanosine Triphosphate/chemistry , Guanosine Triphosphate/metabolism , Hydrolysis , Kinetics , Peptide Termination Factors/chemistry , Peptide Termination Factors/metabolism , Protein Binding , Protein Conformation , RNA, Transfer, Amino Acyl/metabolism , Ribosomes/metabolism , Single Molecule Imaging , ThermodynamicsABSTRACT
The ribosome stalls on translation of polyproline sequences due to inefficient peptide bond formation between consecutive prolines. The translation factor EF-P is able to alleviate this stalling by accelerating Pro-Pro formation. However, the mechanism by which EF-P recognizes the stalled complexes and accelerates peptide bond formation is not known. Here, we use genetic code reprogramming through a flexible in-vitro translation (FIT) system to investigate how mutations in tRNA(Pro) affect EF-P function. We show that the 9-nt D-loop closed by the stable D-stem sequence in tRNA(Pro) is a crucial recognition determinant for EF-P. Such D-arm structures are shared only among the tRNA(Pro) isoacceptors and tRNA(fMet) in Escherichia coli, and the D-arm of tRNA(fMet) is essential for EF-P-induced acceleration of fMet-puromycin formation. Thus, the activity of EF-P is controlled by recognition elements in the tRNA D-arm.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/physiology , Peptide Elongation Factors/metabolism , Protein Biosynthesis , RNA, Transfer, Pro/metabolism , Binding Sites/genetics , Escherichia coli Proteins/genetics , Mutation , Nucleotide Motifs/genetics , Peptide Elongation Factors/genetics , Peptides/metabolism , Protein Binding/genetics , Puromycin/chemistry , Puromycin/metabolism , RNA, Transfer, Met/chemistry , RNA, Transfer, Met/metabolism , RNA, Transfer, Pro/chemistry , RNA, Transfer, Pro/genetics , Ribosomes/genetics , Ribosomes/metabolismABSTRACT
Elongation factor P (EF-P) is a translation factor of unknown function that has been implicated in a great variety of cellular processes. Here, we show that EF-P prevents ribosome from stalling during synthesis of proteins containing consecutive prolines, such as PPG, PPP, or longer proline strings, in natural and engineered model proteins. EF-P promotes peptide-bond formation and stabilizes the peptidyl-transfer RNA in the catalytic center of the ribosome. EF-P is posttranslationally modified by a hydroxylated ß-lysine attached to a lysine residue. The modification enhances the catalytic proficiency of the factor mainly by increasing its affinity to the ribosome. We propose that EF-P and its eukaryotic homolog, eIF5A, are essential for the synthesis of a subset of proteins containing proline stretches in all cells.
Subject(s)
Escherichia coli/metabolism , Peptide Elongation Factors/metabolism , Proline/metabolism , Ribosomes/metabolism , Amino Acid Sequence , Escherichia coli/genetics , Lysine/metabolism , Molecular Sequence Data , Proline/genetics , Protein Biosynthesis , Protein Processing, Post-TranslationalABSTRACT
The emergence of ribosomes and translation factors is central for understanding the origin of life. Recruitment of translation factors to bacterial ribosomes is mediated by the L12 stalk composed of protein L10 and several copies of protein L12, the only multi-copy protein of the ribosome. Here we predict stoichiometries of L12 stalk for >1,200 bacteria, mitochondria and chloroplasts by a computational analysis, and validate the predictions by quantitative mass spectrometry. The majority of bacteria have L12 stalks allowing for binding of four or six copies of L12, largely independent of the taxonomic group or living conditions of the bacteria, whereas some cyanobacteria have eight copies. Mitochondrial and chloroplast ribosomes can accommodate six copies of L12. The last universal common ancestor probably had six molecules of L12 molecules bound to L10. Changes of the stalk composition provide a unique possibility to trace the evolution of protein components of the ribosome.
Subject(s)
Bacteria/metabolism , Bacterial Proteins/genetics , Evolution, Molecular , Ribosomal Proteins/genetics , Ribosomes/metabolism , Amino Acid Sequence , Bacteria/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Chloroplasts/metabolism , Gene Dosage , Humans , Mass Spectrometry , Mitochondria/metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Molecular Sequence Data , Phylogeny , Protein Binding , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Ribosomal, 16S/genetics , Ribosomal Protein L10 , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Synechococcus/metabolism , Thermotoga maritima/genetics , Thermotoga maritima/metabolismABSTRACT
Speed and accuracy of protein synthesis are fundamental parameters for the fitness of living cells, the quality control of translation, and the evolution of ribosomes. The ribosome developed complex mechanisms that allow for a uniform recognition and selection of any cognate aminoacyl-tRNA (aa-tRNA) and discrimination against any near-cognate aa-tRNA, regardless of the nature or position of the mismatch. This review describes the principles of the selection-kinetic partitioning and induced fit-and discusses the relationship between speed and accuracy of decoding, with a focus on bacterial translation. The translational machinery apparently has evolved towards high speed of translation at the cost of fidelity.
Subject(s)
Evolution, Molecular , Protein Biosynthesis , Ribosomes/genetics , Bacterial Proteins/chemistry , Catalytic Domain , Codon/chemistry , Enzyme Activation , Escherichia coli/chemistry , Escherichia coli/genetics , GTP Phosphohydrolases/chemistry , Guanosine Triphosphate/chemistry , Hydrolysis , Kinetics , Peptide Elongation Factor Tu/chemistry , Peptides/chemistry , RNA, Transfer, Amino Acyl/chemistry , Ribosomes/chemistry , Time FactorsABSTRACT
The ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s(-1), regardless of the tRNA identity. The reactivity decreased in the order Lys = Arg > Ala > Ser > Phe = Val > Asp >> Pro, with Pro being by far the slowest. However, when Phe-tRNA(Phe) was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was approximately 7 s(-1), which corresponds to the rate of binding of Phe-tRNA(Phe) to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyl-tRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation.
Subject(s)
RNA, Transfer/chemistry , Amino Acids/chemistry , Catalysis , Codon , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Kinetics , Nucleic Acid Conformation , Peptides/chemistry , Proline/chemistry , Protein Structure, Tertiary , RNA, Catalytic/chemistry , Ribosomes/chemistry , Substrate Specificity , ThermodynamicsABSTRACT
The catalytic site of the ribosome, the peptidyl transferase centre, is located on the large (50S in bacteria) ribosomal subunit. On the basis of results obtained with small substrate analogues, isolated 50S subunits seem to be less active in peptide bond formation than 70S ribosomes by several orders of magnitude, suggesting that the reaction mechanisms on 50S subunits and 70S ribosomes may be different. Here we show that with full-size fMet-tRNA(fMet) and puromycin or C-puromycin as peptide donor and acceptor substrates, respectively, the reaction proceeds as rapidly on 50S subunits as on 70S ribosomes, indicating that the intrinsic activity of 50S subunits is not different from that of 70S ribosomes. The faster reaction on 50S subunits with fMet-tRNA(fMet), compared with oligonucleotide substrate analogues, suggests that full-size transfer RNA in the P site is important for maintaining the active conformation of the peptidyl transferase centre.