Importance of tRNA interactions with 23S rRNA for peptide bond formation on the ribosome: studies with substrate analogs

The major enzymatic activity of the ribosome is the catalysis of peptide bond formation. The active site -- the peptidyl transferase center -- is composed of ribosomal RNA (rRNA), and interactions between rRNA and the reactants, peptidyl-tRNA and aminoacyl-tRNA, are crucial for the reaction to proceed rapidly and efficiently. Here, we describe the influence of rRNA interactions with cytidine residues in A-site substrate analogs (C-puromycin or CC-puromycin), mimicking C74 and C75 of tRNA on the reaction. Base-pairing of C75 with G2553 of 23S rRNA accelerates peptide bond formation, presumably by stabilizing the peptidyl transferase center in its productive conformation. When C74 is also present in the substrate analog, the reaction is slowed down considerably, indicating a slow step in substrate binding to the active site, which limits the reaction rate. The tRNA-rRNA interactions lead to a robust reaction that is insensitive to pH changes or base substitutions in 23S rRNA at the active site of the ribosome.     

The peptidyltransferase centre of the Escherichia coli ribosome. The histidine of protein L16 affects the reconstitution and control of the active centre but is not essential for release-factor-mediated peptidyl-tRNA hydrolysis and peptide bond formation

Modification of the Escherichia coli 50S ribosomal subunit with histidine-specific diethyl pyrocarbonate affects peptide bond formation and release-factor-dependent peptidyl-tRNA hydrolysis. Unmodified L16 can restore activity to a split protein fraction from the altered subunit but other proteins of the core also contain histidine residues important for the activity of the peptidyltransferase centre. When isolated and purified by centrifugation, particles reconstituted with unmodified proteins and modified L16 do not retain the altered L16. The modified protein does mediate the partial restoration of peptide bond formation and release-factor-2 activities to these particles. It must be exerting its effect during the assembly of the peptidyltransferase centre in the reconstituted particle. A particle could be reconstituted which lacks L16 and has significant activity in peptide bond formation and peptidyl-tRNA hydrolysis. L16 stimulates these activities. A tighter ribosomal binding of the release factor 2, dependent upon the absence of protein L11, can in part compensate for the loss of activity of the peptidyltransferase centre when it is assembled with either modified L16 or in the absence of L16. The protein and its histidine residue seem important, therefore, for the peptidyltransferase centre to be formed in the correct conformation but not essential for activity once the centre is assembled.     

Peptide bond formation stimulated by protein synthesis factor EF-P depends on the aminoacyl moiety of the acceptor.

Elongation factor EF-P is a soluble protein that stimulates peptide bond synthesis catalyzed by the 50-S ribosomal subunit. This factor was previously identified and characterized based on its ability to promote the synthesis of formylmethionine-puromycin. In the present work, we tested the ability of EF-P to promote peptide bond synthesis between ribosome-bound fMet-tRNA and several analogues of the 3' terminus of aminoacyl-tRNA, i.e. the cytidylyl(3'-5')-[2'(3')-O-L-aminoacyladenosines]. EF-P promoted synthesis to the greatest extent with certain acceptors which were otherwise inefficient in the peptidyl transferase reaction. This activity of EF-P could not be replaced by the other soluble proteins known to be involved in polypeptide synthesis, such as EF-Tu, EF-Ts and EF-G. One role of EF-P in protein synthesis may be to allow peptide bond synthesis to occur more efficiently with some aminoacyl-tRNAs that are poor acceptors for the ribosomal peptidyl transferase.     

Modification of histidine residues on proteins from the 50S subunit of the Escherichia coli ribosome. Effects on subunit assembly and peptidyl transferase centre activity.

L2, L3, L4, L16 and L20 are proteins of the 50S ribosomal subunit of Escherichia coli which are essential for the assembly and activity of the peptidyl transferase centre. These proteins have been modified with the histidine-specific reagent, diethylpyrocarbonate, while L17 and L18 were treated as controls. Each modified protein tested was able to participate in the reconstitution of a 50S particle when replacing its normal counterpart, although the particles assembled with modified L2 were heterogeneous. However, although they could support assembly, modified L16 and L20 were not themselves reconstituted stably, and modified L2 and L3 were found in less than stoichiometric amounts. Particles assembled in the presence of modified L16 retained significant peptidyl transferase activity (60-70% at 10 mM diethylpyrocarbonate) whereas those reconstituted with modified L2, L3, L4 or L20 had low activity (10-30% at 10 mM diethylpyrocarbonate). The particles assembled with the modified control protein, L17, retained 80% of their peptidyl transferase activity under the same conditions. The histidine residues within the essential proteins therefore contribute to ribosome structure and function in three significant ways; in the correct assembly of the ribosomal subunit (L2), for the stable assembly of the proteins within the ribosomal particle (L20 and L16 in particular), and directly or indirectly for the subsequent activity of the peptidyl transferase centre (L2, L3, L4 and L20). The essential nature of the unmodified histidines for assembly events precludes the use of the chemical-modification strategy to test the proposal that a histidine on one of the proteins might participate in the catalytic activity of the centre.     

SPARK--a novel method to monitor ribosomal peptidyl transferase activity

The key enzymatic activity of the ribosome is catalysis of peptide bond formation. This reaction is a target for many clinically important antibiotics. However, the molecular mechanisms of the peptidyl transfer reaction, the catalytic contribution of the ribosome, and the mechanisms of antibiotic action are still poorly understood. Here we describe a novel, simple, convenient, and sensitive method for monitoring peptidyl transferase activity (SPARK). In this method, the ribosomal peptidyl transferase forms a peptide bond between two ligands, one of which is tritiated whereas the other is biotin-tagged. Transpeptidation results in covalent attachment of the biotin moiety to a tritiated compound. The amount of the reaction product is then directly quantified using the scintillation proximity assay technology: binding of the tritiated radioligand to the commercially available streptavidin-coated beads causes excitation of the bead-embedded scintillant, resulting in detection of radioactivity. The reaction is readily inhibited by known antibiotics, inhibitors of peptide bond formation. The method we developed is amenable to simple automation which makes it useful for screening for new antibiotics. The method is useful for different types of ribosomal research. Using this method, we investigated the effect of mutations at a universally conserved nucleotide of the active site of 23S rRNA, A2602 (Escherichia coli numbering), on the peptidyl transferase activity of the ribosome. The activities of the in vitro reconstituted mutant subunits, though somewhat reduced, were comparable with those of the subunits assembled with the wild-type 23S rRNA, indicating that A2602 mutations do not abolish the ability of the ribosome to catalyze peptide bond formation. Similar results were obtained with double mutants carrying mutations at A2602 and another universally conserved nucleotide in the peptidyl transferase center, A2451. The obtained results agree with our previous conclusion that the ribosome accelerates peptide bond formation primarily through entropic rather than chemical catalysis.     

An alternative mechanism for the catalysis of peptide bond formation by L/F transferase: substrate binding and orientation

Eubacterial leucyl/phenylalanyl tRNA protein transferase (L/F transferase) catalyzes the transfer of a leucine or a phenylalanine from an aminoacyl-tRNA to the N-terminus of a protein substrate. This N-terminal addition of an amino acid is analogous to that of peptide synthesis by ribosomes. A previously proposed catalytic mechanism for Escherichia coli L/F transferase identified the conserved aspartate 186 (D186) and glutamine 188 (Q188) as key catalytic residues. We have reassessed the role of D186 and Q188 by investigating the enzymatic reactions and kinetics of enzymes possessing mutations to these active-site residues. Additionally three other amino acids proposed to be involved in aminoacyl-tRNA substrate binding are investigated for comparison. By quantitatively measuring product formation using a quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based assay, our results clearly demonstrate that, despite significant reduction in enzymatic activity as a result of different point mutations introduced into the active site of L/F transferase, the formation of product is still observed upon extended incubations. Our kinetic data and existing X-ray crystal structures result in a proposal that the critical roles of D186 and Q188, like the other amino acids in the active site, are for substrate binding and orientation and do not directly participate in the chemistry of peptide bond formation. Overall, we propose that L/F transferase does not directly participate in the chemistry of peptide bond formation but catalyzes the reaction by binding and orientating the substrates for reaction in an analogous mechanism that has been described for ribosomes.     

Functional roles of 50-S ribosomal proteins.

Ribosomal proteins previously inactivated by treatment with fluorescein isothiocyanate have been incorporated into 50-S ribosomal subunits during reconstitution from particles disassembled by 2 M LiCl in the presence of an excess of the modified proteins. The reconstituted particles show alterations in some functional activities resulting from the incorporation of the inactive ribosomal proteins added exogenously. Of the fluorescein-isothiocyanate-treated proteins incorporated, L24 and L25 drastically affect all the activities tested and these proteins possibly play a fundamental role in determining the overall structure of the particle. Proteins L16 and L10 are apparently involved both in the GTP hydrolysis dependent on elongation factor G and in peptidyl transferase activity but the modified protein L11 only affects GTPase activity indirectly and interferes with the ribosome assembly process involving proteins L7 and L12. Protein L1 may be involved with peptidyl transferase activity while proteins L7 and L12, in agreement with many reports in the literature, affect the factor-dependent hydrolysis of GTP.     

Changes in ribosome function induced by protein kinase associated with ribosomes of Streptomyces collinus producing kirromycin.

Protein kinase associated with ribosomes of streptomycetes phosphorylates 11 ribosomal proteins. Phosphorylation activity of protein kinase reaches its maximum at the end of exponential phase of growth. When (32)P-labeled cells from the end of exponential phase of growth were transferred to a fresh medium, after 2 h of cultivation ribosomal proteins lost more than 90% of (32)P and rate of polypeptide synthesis increases twice. Protein kinase cross-reacting with antibody raised against protein kinase C was partially purified from 1 M NH(4)Cl wash of ribosomes and used to phosphorylation of ribosomes. Phosphorylation of 50S subunits (L2, L3, L7, L16, L21, L23, and L27) had no effect on the integrity of subunits but affects association with 30 to 70S monosomes. In vitro system derived from ribosomal subunits was used to examine the activity of phosphorylated 50S at poly(U) translation. Replacement unphosphorylated 50S with 50S possessed of phosphorylated r-proteins leads to the reduction of polypeptide synthesis of about 52%. The binding of N-Ac[(14)C]Phe-tRNA to A-site of phosphorylated ribosomes is not affected but the rate of peptidyl transferase is more than twice lower than that in unphosphorylated ribosomes. These results provide evidence that phosphorylation of ribosomal proteins is involved in mechanisms regulating the translational system of Streptomyces collinus.     

The ribosomal peptidyl transferase center: structure, function, evolution, inhibition

The ribosomal peptidyl transferase center (PTC) resides in the large ribosomal subunit and catalyzes the two principal chemical reactions of protein synthesis: peptide bond formation and peptide release. The catalytic mechanisms employed and their inhibition by antibiotics have been in the focus of molecular and structural biologists for decades. With the elucidation of atomic structures of the large ribosomal subunit at the dawn of the new millennium, these questions gained a new level of molecular significance. The crystallographic structures compellingly confirmed that peptidyl transferase is an RNA enzyme. This places the ribosome on the list of naturally occurring ribozymes that outlived the transition from the pre-biotic RNA World to contemporary biology. Biochemical, genetic and structural evidence highlight the role of the ribosome as an entropic catalyst that accelerates peptide bond formation primarily by substrate positioning. At the same time, peptide release should more strongly depend on chemical catalysis likely involving an rRNA group of the PTC. The PTC is characterized by the most pronounced accumulation of universally conserved rRNA nucleotides in the entire ribosome. Thus, it came as a surprise that recent findings revealed an unexpected high level of variation in the mode of antibiotic binding to the PTC of ribosomes from different organisms.     

The modification of the peptidyl transferase activity of 50-S ribosomal subunits, LiC1-split proteins and L16 ribosomal protein by ethoxyformic anhydride.

Ethoxyformic anhydride abolishes the peptidyl transferase activity of 50-S ribosomal subunits, LiC1 split proteins and L16. Hydroxylamine treatment results in reactivation. Erythromycin exhibits significant protection with 50-S ribosomal subunits. With LiC1 split proteins and L16 significant protection was exhibited only after reconstitution. The results indicate that the ethoxyformic anhydride is reacting with approximately six histidines in LiC1 split proteins and one in L16. Since L16 has been reported to contain a single histidine, the results presented indicate the involvement of this histidine in peptidyl transferase activity.     

The Ribosomal Peptidyl Transferase Center: Structure,Function, Evolution,Inhibition

ABSTRACT

The ribosomal peptidyl transferase center (PTC) resides in the large ribosomal subunit and catalyzes the two principal chemical reactions of protein synthesis: peptide bond formation and peptide release. The catalytic mechanisms employed and their inhibition by antibiotics have been in the focus of molecular and structural biologists for decades. With the elucidation of atomic structures of the large ribosomal subunit at the dawn of the new millennium, these questions gained a new level of molecular significance. The crystallographic structures compellingly confirmed that peptidyl transferase is an RNA enzyme. This places the ribosome on the list of naturally occurring riboyzmes that outlived the transition from the pre-biotic RNA World to contemporary biology. Biochemical, genetic and structural evidence highlight the role of the ribosome as an entropic catalyst that accelerates peptide bond formation primarily by substrate positioning. At the same time, peptide release should more strongly depend on chemical catalysis likely involving an rRNA group of the PTC. The PTC is characterized by the most pronounced accumulation of universally conserved rRNA nucleotides in the entire ribosome. Thus, it came as a surprise that recent findings revealed an unexpected high level of variation in the mode of antibiotic binding to the PTC of ribosomes from different organisms.     

Phosphorylation of ribosomal proteins influences subunit association and translation of poly (U) in Streptomyces coelicolor

The occurrence of phosphorylated proteins in ribosomes of Streptomyces coelicolor was investigated. Little is known about which biological functions these posttranslational modifications might fulfil. A protein kinase associated with ribosomes phosphorylated six ribosomal proteins of the small subunit (S3, S4, S12, S13, S14 and S18) and seven ribosomal proteins of the large subunit (L2, L3, L7/L12, L16, L17, L23 and L27). The ribosomal proteins were phosphorylated mainly on the Ser/Thr residues. Phosphorylation of the ribosomal proteins influences ribosomal subunits association. Ribosomes with phosphorylated proteins were used to examine poly (U) translation activity. Phosphorylation induced about 50% decrease in polyphenylalanine synthesis. After preincubation of ribosomes with alkaline phosphatase the activity of ribosomes was greatly restored. Small differences were observed between phosphorylated and unphosphorylated ribosomes in the kinetic parameters of the binding of Phe-tRNA to the A-site of poly (U) programmed ribosomes, suggesting that the initial binding of Phe-tRNA is not significantly affected by phosphorylation. On contrary, the rate of peptidyl transferase was about two-fold lower than that in unphosphorylated ribosomes. The data presented demonstrate that phosphorylation of ribosomal proteins affects critical steps of protein synthesis.     

Reactivity of the P-site-bound donor in ribosomal peptide-bond formation

The puromycin reaction, catalyzed by the ribosomal peptidyltransferase, has been carried out so as to make the definition of two distinct parameters of this reaction possible. These are (a) the final degree of the reaction which gives the proportion of peptidyl (P)-site binding of the donor and (b) the reactivity of the donor substrate expressed as an apparent rate constant (kobs). This kobs varies with the concentration of puromycin; the maximal value (k3) of the kobs, at saturating concentrations of puromycin, gives the reactivity of the donor independently of the concentrations of both the donor and puromycin. k3 is also a measure of the activity of peptidyltransferase expressed as its catalytic rate constant (kcat). If we assume that the puromycin-reactive donor is bound at the ribosomal P site, we observe the following, depending on the conditions of the experiment: the proportion of P-site binding of the donor substrates AcPhe-tRNA or fMet-tRNA can be the same and close to 100%, while there is a tenfold increase in the reactivity of the donor (k3 = 0.8 min-1 versus 8.3 min-1). On the other hand there are conditions, under which the proportion of P-site binding increases from 30% to 100% while k3 remains low and equal to 0.8 min-1. Using the puromycin reaction it was also found that an increase of Mg2+ from 10 mM to 20 mM reduces the reactivity of the donor and, hence, the activity of peptidyltransferase, provided that this change in Mg2+ occurs during the binding of the donor but not when it occurs during peptide bond formation per se. The fact that the donor substrate may exist in various states of reactivity in this cell-free system raises the possibility that the rate of peptide bond formation may not be uniform during protein synthesis.     

Influence of cholesteryl 14-methylhexadecanoate on some ribosomal functions required for peptide elongation

1. Polyribosomes and ribosomal subunits from rat liver were adsorbed on a cellulosic ion-exchange adsorbent, freeze-dried and extracted with organic solvents. The activity of extracted particles in peptide elongation was tested in the presence of purified peptideelongation factors. 2. Chloroform-methanol mixture (2:1, v/v) extracted 1.87+/-0.15 pmol of cholesteryl 14-methylhexadecanoate/pmol of the smaller ribosomal subunit and 0.92+/-0.11 pmol/pmol of the larger subunit. 3. In the presence of transferase I, extracted polyribosomes and 40S subunits bound more phenylalanyl-tRNA than did control non-extracted particles. The same binding as in control mixtures was obtained with extracted particles supplemented with cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. The polymerization of phenylalanine was greatly decreased with extracted polyribosomes and subunits and addition of the cholesteryl ester could not fully restore the original activity. 5. Extraction significantly decreased the activity of the P site of peptidyl transferase and normal activity was recovered after the addition of the ester. The A site of peptidyl transferase in extracted polyribosomes showed an increased activity when compared with non-extracted polyribosomes. 6. Cholesteryl 14-methylhexadecanoate apparently affects the function of the ribosomal A site and peptidyl transferase site and probably also that of the guanosine triphosphatase site and P site. The presence of different amounts of the ester in polyribosomes may be one of the mechanisms modulating peptide elongation at the ribosomal level.     

Structural basis for the inability of chloramphenicol to inhibit peptide bond formation in the presence of A-site glycine

Ribosome serves as a universal molecular machine capable of synthesis of all the proteins in a cell. Small-molecule inhibitors, such as ribosome-targeting antibiotics, can compromise the catalytic versatility of the ribosome in a context-dependent fashion, preventing transpeptidation only between particular combinations of substrates. Classic peptidyl transferase center inhibitor chloramphenicol (CHL) fails to inhibit transpeptidation reaction when the incoming A site acceptor substrate is glycine, and the molecular basis for this phenomenon is unknown. Here, we present a set of high-resolution X-ray crystal structures that explain why CHL is unable to inhibit peptide bond formation between the incoming glycyl-tRNA and a nascent peptide that otherwise is conducive to the drug action. Our structures reveal that fully accommodated glycine residue can co-exist in the A site with the ribosome-bound CHL. Moreover, binding of CHL to a ribosome complex carrying glycyl-tRNA does not affect the positions of the reacting substrates, leaving the peptide bond formation reaction unperturbed. These data exemplify how small-molecule inhibitors can reshape the A-site amino acid binding pocket rendering it permissive only for specific amino acid residues and rejective for the other substrates extending our detailed understanding of the modes of action of ribosomal antibiotics.     

Toward Ribosomal RNA Catalytic Activity in the Absence of Protein

The ribosome is the ribonucleoprotein particle responsible for translation of genetic information into proteins. The RNA component of the ribosome has been implicated as the catalytic entity for peptide bond formation based on protease resistance and structural data indicating an all-RNA active site. Nevertheless, peptidyl transfer by ribosomal RNA (rRNA) alone has not been demonstrated. In an attempt to show such activity we generated a minimal construct that comprises much of the 23S rRNA peptidyl transferase center, including the central loop and the A- and P-loops. This minimal rRNA domain was inactive in peptide bond formation under all conditions tested. The RNA was subsequently subjected to six rounds of in vitro selection designed to enrich for this activity. The result was a mutated rRNA sequence that could catalyze the covalent linkage of an A-site and P-site substrate; however, the product did not contain a peptide bond. The current study is an example of an in vitro derived alternate function of rRNA mutants and illustrates the evolutionary possibility that the protoribosome may have used amino acids as substrates before it gained the ability to join them into peptides. Though peptidyl transferase activity in the absence of protein remains elusive, the ease with which alternate catalytic activity was selected from rRNA with a small number of mutations suggests that rRNA may have inherent activity. This study represents a step on the path toward isolating that native activity. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. Niles Lehman]     

Chemical inactivation of Escherichia coli 30-S ribosomes by iodination. Identification of proteins involved in tRNA binding.

30-S ribosomal subunits are inactivated by iodination for both enzymic fMet-tRNA and non-enzymic Phe-tRNA binding activities. This inactivation is due to modification of the protein moiety of the ribosome. Reconstitutions were performed with 16-S RNA and mixtures of total protein isolated from modified subunits and purified proteins isolated from unmodified subunits. This allowed identification of the individual proteins which restore tRNA binding activity. S3, S14 and S19 were identified as proteins involved in fMet-tRNA binding. S1, S2, S3, S14 and S19 were identified as proteins involved in Phe-tRNA binding. Modified particles shown normal sedimentation constants and complete protein compositions both before and after reconstitution. This suggests that the loss of activity is due to modification of one or more of the actual binding sites located on the 30-S subunit and that restoration of activity is due to structural correction at this site rather than to correction of an assembly defect.     

The composition and properties of the Escherichia coli 5-S RNA-protein complex

At a high concentration of MgCl2 (30 mM) and a low concentration of proteins from the 50-S subunit (0.2 mg/ml), only three proteins, L15, L18 and L25, bind to 5-S RNA in significant amounts. On the other hand, in a buffer containing only 1 mM Mg Cl2, but otherwise at the same ionic strength (0.2 M), or at a protein concentration about 1.5 mg/ml, a large, stable complex can form between immobilized 5-S RNA and 50-S ribosomal proteins. This complex contains proteins L2, L3, L5, L15, L16, L17, L18, L21, L22, L25, L33 and L34, and it possess properties relevant to the function of the 50-S subunit; it has a binding site for deacylated tRNA, with a dissociation constant of 4.5 x 10(-7) M. The complex formed with 5-S RNA immobilized on an affinity column interacts also with 30-S subunits. The 5-S RNA-protein complex is interpreted as a sub-ribosomal domain which includes a considerable fraction of the peptidyl transferase center of the Escherichia coli ribosome.     

A functional connection between translation elongation and protein folding at the ribosome exit tunnel in Saccharomyces cerevisiae

Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5′ region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel.     

Structural insights into the roles of water and the 2' hydroxyl of the P site tRNA in the peptidyl transferase reaction

Peptide bond formation is catalyzed at the peptidyl transferase center (PTC) of the large ribosomal subunit. Crystal structures of the large ribosomal subunit of Haloarcula marismortui (Hma) complexed with several analogs that represent either the substrates or the transition state intermediate of the peptidyl transferase reaction show that this reaction proceeds through a tetrahedral intermediate with S chirality. The oxyanion of the tetrahedral intermediate interacts with a water molecule that is positioned by nucleotides A2637 (E. coli numbering, 2602) and (methyl)U2619(2584). There are no Mg2+ ions or monovalent metal ions observed in the PTC that could directly promote catalysis. The A76 2' hydroxyl of the peptidyl-tRNA is hydrogen bonded to the alpha-amino group and could facilitate peptide bond formation by substrate positioning and by acting as a proton shuttle between the alpha-amino group and the A76 3' hydroxyl of the peptidyl-tRNA.