Peptidyl transferase centres of rat and yeast ribosomes. Different response to modification of protein amino groups

Modification of rat liver ribosomes with dimethylmaleic anhydride, a reagent for protein amino groups, causes a large stimulation of peptidyl transferase activity assayed by the "fragment" reaction, as well as the inactivation of poly(U)-directed polyphenylalanine synthesis. In contrast to rat ribosomes, the peptidyl transferase of yeast ribosomes is little affected by modification. Although other interpretations are not excluded, these results might be due to differences between the peptidyl transferase centres of mammalian and yeast ribosomes.     

Role for the highly conserved region of domain IV of 23S-like rRNA in subunit-subunit interactions at the peptidyl transferase centre.

The function of the highly conserved and accessible region of domain IV of 23S rRNA (positions 1900-1981 in Escherichia coli 23S rRNA) was investigated by subjecting it to a random mutagenesis procedure that produced single-site mutations efficiently. Nine single-site mutants were selected that were recessive lethal. High levels of mutated 23S rRNA were expressed in E. coli and extracted ribosomes were investigated for their content of mutated rRNA. The peptidyl transferase activity of the ribosomes was also estimated using a newly developed method involving selective inhibition of chromosome-encoded ribosomes by clindamycin. Two of the mutants, U1940A and U1955G, yielded 50S subunits that were defective in subunit-subunit association but active in peptidyl transferase activity and five, U1926C, U1946C, U1979C, U1982A and G1984A, produced 50S subunits that were defective in both subunit-subunit interactions and peptidyl transferase activity. We infer that the large conserved rRNA region generates a complex structure that plays an essential role in maintaining and modulating subunit-subunit interactions and argue that its involvement in the peptidyl transferase centre is secondary, possibly involving the correct alignment of protein L2.     

Peptidyl transferase of bacterial ribosome: resistance to proteinase K.

70-S ribosomes and 50-S ribosomal subunits from Escherichia coli D10 were treated with proteinase K for increasing periods of time. Peptidyl transferase activity and sparsomycin-induced binding of (U)C-A-C-C-A-[3H]Leu-Ac were tested in the treated particles, the binding of the substrate being more sensitive to the protease than peptide bond formation. Comparison of the amounts of proteins present in the treated particles with the residual activity indicates that only proteins L3 and L14 are released at a similar rate to that at which peptidyl transferase activity is lost. Proteins related to this ribosomal activity by other techniques are lost at a faster rate than the activity itself. In addition, the results indicate that sparsomycin stimulates the binding of the substrate by a different mechanism from that which inhibits peptide bond formation.     

Immunological studies of the fragments of bovine cartilage proteoglycan produced by chondroitinase-trypsin digestion.

The peptidyl transferase activity of polysomes from Escherichia coli, rabbit reticulocytes and chick embryos, assayed in the fragment reaction, is 3- to 10-fold lower than the corresponding activity of single ribosomes. The polysomal peptidyl transferase activity is restored in full under conditions of in vitro protein synthesis that result in conversion of polysomes to single ribosomes. Thus, the peptidyl transferase center is masked in translating ribosomes. Unmasking of peptidyl transferase, however, does not require the release of ribosomes from messenger RNA: it is also seen upon treatment of polysomes with puromycin, under conditions in which polysomes remain intact. Apparently, release of nascent polypeptide chains is sufficient to allow access of formylmethionyl hexanucleotide substrate to the peptidyl transferase site.     

An apparent conformational change in tRNA(Phe) that is associated with the peptidyl transferase reaction

Fluorescence techniques were used to detect changes in the conformation of tRNA(Phe) that may occur during the peptidyl transferase reaction in which the tRNA appears to move between binding sites on ribosomes. Such a conformational change may be a fundamental part of the translocation mechanism by which tRNA and mRNA are moved through ribosomes. E. coli tRNA(Phe) was specifically labeled on acp3U47 and s4U8 or at the D positions 16 and 20. The labeled tRNAs were bound to ribosomes as deacylated tRNA(Phe) or AcPhe-tRNA. Changes in fluorescence quantum yield and anisotropy were measured upon binding to the ribosomes and during the peptidyl transferase reaction. In one set of experiments non-radiative energy transfer was measured between a coumarin probe at position 16 or 20 and a fluorescein attached to acp3U47 on the same tRNA(Phe) molecule. The results indicate that the apparent distance between the probes increases during deacylation of AcPhe-tRNA as a result of peptide bond formation. All of the results are consistent with but in themselves do not conclusively establish that tRNA undergoes a conformational change as well as movement during the peptidyl transferase reaction.     

Identification of proteins involved in the peptidyl transferase activity of ribosomes by chemical modification.

Photochemical oxidation of Escherichia coli 50 S ribosomal subunits in the presence of methylene blue or Rose Bengal causes rapid loss of peptidyl transferase activity. Reconstitution experiments using mixtures of components from modified and unmodified ribosomes reveal that both RNA and proteins are affected, and that among the proteins responsible for inactivation there are both LiCl-split and core proteins. The proteins L2 and L16 from the split fraction and L4 from the core fraction of unmodified ribosomes were together nearly as effective as total unmodified proteins in restoring peptidyl transferase activity to reconstituted ribosomes when added with proteins from modified ribosomes. These three proteins are therefore the most important targets identified as responsible for loss of peptidyl transferase activity on photo-oxidation of 50 S ribosomal subunits.     

The pleuromutilin drugs tiamulin and valnemulin bind to the RNA at the peptidyl transferase centre on the ribosome

The pleuromutilin antibiotic derivatives, tiamulin and valnemulin, inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria. The action and binding site of tiamulin and valnemulin was further characterized on Escherichia coli ribosomes. It was revealed that these drugs are strong inhibitors of peptidyl transferase and interact with domain V of 23S RNA, giving clear chemical footprints at nucleotides A2058-9, U2506 and U2584-5. Most of these nucleotides are highly conserved phylogenetically and functionally important, and all of them are at or near the peptidyl transferase centre and have been associated with binding of several antibiotics. Competitive footprinting shows that tiamulin and valnemulin can bind concurrently with the macrolide erythromycin but compete with the macrolide carbomycin, which is a peptidyl transferase inhibitor. We infer from these and previous results that tiamulin and valnemulin interact with the rRNA in the peptidyl transferase slot on the ribosomes in which they prevent the correct positioning of the CCA-ends of tRNAs for peptide transfer.     

Involvement of mitochondrial ribosomal proteins in ribosomal RNA-mediated protein folding

The peptidyl transferase center of the domain V of large ribosomal RNA in the prokaryotic and eukaryotic cytosolic ribosomes acts as general protein folding modulator. We showed earlier that one part of the domain V (RNA1 containing the peptidyl transferase loop) binds unfolded protein and directs it to a folding competent state (FCS) that is released by the other part (RNA2) to attain the folded native state by itself. Here we show that the peptidyl transferase loop of the mitochondrial ribosome releases unfolded proteins in FCS extremely slowly despite its lack of the rRNA segment analogous to RNA2. The release of FCS can be hastened by the equivalent activity of RNA2 or the large subunit proteins of the mitochondrial ribosome. The RNA2 or large subunit proteins probably introduce some allosteric change in the peptidyl transferase loop to enable it to release proteins in FCS.     

Base-pairing between 23S rRNA and tRNA in the ribosomal A site

The aminoacyl (A site) tRNA analog 4-thio-dT-p-C-p-puromycin (s4TCPm) photochemically cross-links with high efficiency and specificity to G2553 of 23S rRNA and is peptidyl transferase reactive in its cross-linked state, establishing proximity between the highly conserved 2555 loop in domain V of 23S rRNA and the universally conserved CCA end of tRNA. To test for base-pairing interactions between 23S rRNA and aminoacyl tRNA, site-directed mutations were made at the universally conserved nucleotides U2552 and G2553 of 23S rRNA in both E. coli and B. stearothermophilus ribosomal RNA and incorporated into ribosomes. Mutations at G2553 resulted in dominant growth defects in E. coli and in decreased levels of peptidyl transferase activity in vitro. Genetic analysis in vitro of U2552 and G2553 mutant ribosomes and CCA end mutant tRNA substrates identified a base-pairing interaction between C75 of aminoacyl tRNA and G2553 of 23S rRNA.     

Inhibition of the ribosomal peptidyl transferase reaction by the mycarose moiety of the antibiotics carbomycin, spiramycin and tylosin

Many antibiotics, including the macrolides, inhibit protein synthesis by binding to ribosomes. Only some of the macrolides affect the peptidyl transferase reaction. The 16-member ring macrolide antibiotics carbomycin, spiramycin, and tylosin inhibit peptidyl transferase. All these have a disaccharide at position 5 in the lactone ring with a mycarose moiety. We have investigated the functional role of this mycarose moiety. The 14-member ring macrolide erythromycin and the 16-member ring macrolides desmycosin and chalcomycin do not inhibit the peptidyl transferase reaction. These drugs have a monosaccharide at position 5 in the lactone ring. The presence of mycarose was correlated with inhibition of peptidyl transferase, footprints on 23 S rRNA and whether the macrolide can compete with binding of hygromycin A to the ribosome. The binding sites of the macrolides to Escherichia coli ribosomes were investigated by chemical probing of domains II and V of 23 S rRNA. The common binding site is around position A2058, while effects on U2506 depend on the presence of the mycarose sugar. Also, protection at position A752 indicates that a mycinose moiety at position 14 in 16-member ring macrolides interact with hairpin 35 in domain II. Competitive footprinting of ribosomal binding of hygromycin A and macrolides showed that tylosin and spiramycin reduce the hygromycin A protections of nucleotides in 23 S rRNA and that carbomycin abolishes its binding. In contrast, the macrolides that do not inhibit the peptidyl transferase reaction bind to the ribosomes concurrently with hygromycin A. Data are presented to argue that a disaccharide at position 5 in the lactone ring of macrolides is essential for inhibition of peptide bond formation and that the mycarose moiety is placed near the conserved U2506 in the central loop region of domain V 23 S rRNA.     

Peptidyl-puromycin synthesis by free and membrane-bound ribosomes.

The peptidyl transferase reaction, as measured by the formation of peptidyl-puromycin, was compared for free ribosomes and ribosomes bound to two types of membrane, the endoplasmic reticulum and the outer nuclear membrane. In most respects the reaction catalyzed by the three types of ribosome was similar, demonstrating that interaction of the 60 S ribosomal subunit with the membrane has little effect on the functioning of peptidyl transferase, a 60 S protein. However, both the rate and extent of synthesis of peptidyl puromycin were lower for ribosomes bound to the nuclear membrane than for free or microsome-bound ribosomes. This difference appears to be a direct consequence of the ribosome-membrane interaction, since ribosomes stripped from the nuclear membrane could not be distinguished from the other classes of ribosome.     

Implication of arginyl residues in aminoacyl-tRNA binding to ribosomes

Modification of the 50-S subunits of Escherichia coli ribosomes with the arginine reagent phenylglyoxal produces inactivation of peptidyl transferase and inhibition of the binding of C(U)-A-C-C-A-LeuAc, phenylalanyl-tRNA and N-acetylphenylalanyl-tRNA to the ribosome. Hybridization experiments, using 1.25 M LiCl core particles and the corresponding split proteins from untreated and phenylglyoxal-treated 50-S subunits, indicate that inactivation and inhibition of binding are the effects of modification of a protein fraction, the functionality of the RNA moiety being unaffected by the reagent. The split proteins from phenylglyoxal-modified 50-S subunits are incorporated to 1.25 M LiCl core particles as well as those obtained from unmodified subunits, thus excluding the failure to bind as the cause of inactivation. In agreement with the general role played by the arginyl residues as positive binding sites for anionic ligands, the present results indicate that the arginyl residues of a protein fraction from 50-S subunits might be important in the binding of aminoacyl-tRNA and peptidyl-tRNA to ribosomes.     

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.     

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.     

MRM2 encodes a novel yeast mitochondrial 21S rRNA methyltransferase

Mitochondria of the yeast Saccharomyces cerevisiae assemble their ribosomes from ribosomal proteins, encoded by the nuclear genome (with one exception), and rRNAs of 15S and 21S, encoded by the mitochondrial genome. Unlike cytoplasmic rRNA, which is highly modified, mitochondrial rRNA contains only three modified nucleotides: a pseudouridine (Psi(2918)) and two 2'-O-methylated riboses (Gm(2270) and Um(2791)) located at the peptidyl transferase centre of 21S rRNA. We demonstrate here that the yeast nuclear genome encodes a mitochondrial protein, named Mrm2, which is required for methylating U(2791) of 21S rRNA, both in vivo and in vitro. Deletion of the MRM2 gene causes thermosensitive respiration and leads to rapid loss of mitochondrial DNA. We propose that Mrm2p belongs to a new class of three eukaryotic RNA-modifying enzymes and is the orthologue of FtsJ/RrmJ, which methylates a nucleotide of the peptidyl transferase centre of Escherichia coli 23S rRNA that is homologous to U(2791) of 21S rRNA. Our data suggest that this universally conserved modified nucleotide plays an important function in vivo, possibly by inducing conformational rearrangement of the peptidyl transferase centre.     

Rapid peptide bond formation on isolated 50S ribosomal subunits

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.     

Transit of tRNA through the Escherichia coli ribosome. Cross-linking of the 3' end of tRNA to specific nucleotides of the 23 S ribosomal RNA at the A, P, and E sites

When bound to Escherichia coli ribosomes and irradiated with near-UV light, various derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at the 3' terminus form cross-links to 23 S rRNA and 50 S subunit proteins in a site-dependent manner. A and P site-bound tRNAs, whose 3' termini reside in the peptidyl transferase center, label primarily nucleotides U2506 and U2585 and protein L27. In contrast, E site-bound tRNA labels nucleotide C2422 and protein L33. The cross-linking patterns confirm the topographical separation of the peptidyl transferase center from the E site domain. The relative amounts of label incorporated into the universally conserved residues U2506 and U2585 depend on the occupancy of the A and P sites by different tRNA ligands and indicates that these nucleotides play a pivotal role in peptide transfer. In particular, the 3'-adenosine of the peptidyl-tRNA analogue, AcPhe-tRNA(Phe), remains in close contact with U2506 regardless of whether its anticodon is located in the A site or P site. Our findings, therefore, modify and extend the hybrid state model of tRNA-ribosome interaction. We show that the 3'-end of the deacylated tRNA that is formed after transpeptidation does not immediately progress to the E site but remains temporarily in the peptidyl transferase center. In addition, we demonstrate that the E site, defined by the labeling of nucleotide C2422 and protein L33, represents an intermediate state of binding that precedes the entry of deacylated tRNA into the F (final) site from which it dissociates into the cytoplasm.     

Photoaffinity labeling of the ribosomal peptidyl transferase site with synthetic puromycin analogues.

A photoaffinity labeling puromycin analogue, Nepsilon-(2-nitro-4-azidophenyl)-L-lysinyl puromycin aminonucleoside (NAP-Lys-Pan), was synthesized and used for investigation of the peptidyl transferase center of 70S riobsomes. Visible light irradiation of NAP-Lys-Pan led to covalent linkage of the analogue with Escherichia coli ribosomes. In a subsequent step, poly(uridylic acid) was employed to direct Ac[14C]Phe-tRNA to the P sites of the photolabeled ribosomes. Transpeptidation of Ac[14C]phenylalanine to the bound NAP-Lys-Pan resulted in selective incorporation of radioactive label into the peptidyl transferase A site. Dissociation of the ribosomes into subunits, and digestion of the RNA components, indicated that the radioactive label was incorporated into a protein fraction of the 50S subunit.     

Ribosome protection by tRNA derivatives against inactivation by virginiamycin M: evidence for two types of interaction of tRNA with the donor site of peptidyl transferase

Virginiamycin M (VM) was previously shown to interfere with the function of both the A and P sites of ribosomes and to inactivate tRNA-free ribosomes but not particles bearing peptidyl-tRNA. To explain these findings, the shielding ability afforded by tRNA derivatives positioned at the A and P sites against VM-produced inactivation was explored. Unacylated tRNA(Phe) was ineffective, irrespective of its position on the ribosome. Phe-tRNA and Ac-Phe-tRNA provided little protection when bound directly to the P site but were active when present at the A site. Protection by these tRNA derivatives was markedly enhanced by the formation of the first peptide bond and increased further upon elongation of peptide chains. Most of the shielding ability of Ac-Phe-tRNA and Phe-tRNA positioned at the A site was conserved when these tRNAs were translocated to the P site by the action of elongation factor G and GTP. Thus, a 5-10-fold difference in the protection afforded by these tRNAs was observed, depending on their mode of entry to the P site. This indicates the occurrence of two types of interaction of tRNA derivatives with the donor site of peptidyl transferase: one shared by acylated tRNAs directly bound to the ribosomal P site (no protection against VM) and the other characteristic of aminoacyl- or peptidyl-tRNA translocated from the A site (protection of peptidyl transferase against VM). To explain these data and previous observations with other protein synthesis inhibitors, a new model of peptidyl transferase is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Puromycin interacts with the donor (P) site of Escherichia coli ribosomes

Puromycin inhibits the interaction of peptidyl-tRNA analogs AcPhe-tRNA Phe ox-red, AcPhe-tRNA Phe and FMet-tRNA f Met with the donor (P) site of Escherichia coli ribosomes. It affects both template-free and poly(U)-dependent systems. The inhibition is apparently due to direct competition for the P-site. On isolated 30S ribosomal subunits it was shown that the puromycin binding site is situated far from the peptidyl transferase center. Quantitative measurements of the inhibition revealed that the affinity constant of puromycin for the P-site is not less than its affinity for the A-moiety of the peptidyl transferase center [1.1 divided by 3.8) X 10(3) M-1).