Effect of cytidine-5'-monophosphate on peptidyl transferase activity.

The transfer reaction with pA-fMet as a donor substrate is strongly stimulated by CMP, whereas the transfer reaction with CpApCpCpA-acLeu as a donor substrate is inhibited by CMP. These results indicate that the donor site of peptidyl transferase contains specific binding sites for the terminal adenosine and for the cytidylic acid residue in the terminal sequence CpCpA of tRNA and that an attachment of proper nucleotides to the donor site induces a conformational change in peptidyl transferase.     

On the molecular mechanism of action of certain substrates and inhibitors of ribosomal peptidyl transferase

Conformational analysis of a number of inhibitors and substrates of Escherichia coli ribosomal peptidyl transferase has indicated that they are analogs of the 3′-terminus of aminoacyl- or peptidyl-tRNA and that their primary locus of action is the acceptor site of this enzyme. The evidence available in the literature which supports the proposed scheme has been reviewed. Specific, experimentally accessible predictions of the scheme are given.     

Puromycin binding to the donor (P-) site of Escherichia coli ribosomes

Puromycin inhibits the interaction of peptidyl-tRNA analogues AcPhe-tRNA_ox-red^Phe, AcPhe-tRNA^Phe and fMet-tRNA_f^Met with the donor (P-) site of Escherichia coli ribosomes. affects almost equally both the rate of the binding and the equilibrium of the system. This means that the effect is due to direct competition for the P-site, but not due to the indirect influence via the acceptor (A-) site. The inhibition was observed also in 30 S ribosomal subunits, therefore the puromycin binding site is situated far from the peptidyl transferase center. Quantitative measurements show that the affinity of puromycin for its new ribosomal binding site is similar to its affinity for the acceptor site of the peptidyl transferase center.     

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)     

The crystal structure of human eukaryotic release factor eRF1--mechanism of stop codon recognition and peptidyl-tRNA hydrolysis

The release factor eRF1 terminates protein biosynthesis by recognizing stop codons at the A site of the ribosome and stimulating peptidyl-tRNA bond hydrolysis at the peptidyl transferase center. The crystal structure of human eRF1 to 2.8 A resolution, combined with mutagenesis analyses of the universal GGQ motif, reveals the molecular mechanism of release factor activity. The overall shape and dimensions of eRF1 resemble a tRNA molecule with domains 1, 2, and 3 of eRF1 corresponding to the anticodon loop, aminoacyl acceptor stem, and T stem of a tRNA molecule, respectively. The position of the essential GGQ motif at an exposed tip of domain 2 suggests that the Gln residue coordinates a water molecule to mediate the hydrolytic activity at the peptidyl transferase center. A conserved groove on domain 1, 80 A from the GGQ motif, is proposed to form the codon recognition site.     

Hygromycin A, a novel inhibitor of ribosomal peptidyltransferase

In cell-free systems from Escherichia coli, hygromycin A inhibits polypeptide synthesis directed by either poly(U) or phage R 17 RNA, and the reaction of puromycin with either natural peptidyl-tRNA, or AcPhe-tRNA, or the 3'-terminal fragment of AcLeu-tRNA (C-A-C-C-A-LeuAc). In contrast, the antibiotic does no inhibit the enzymatic binding of Phe-tRNA to ribosomes or the translocation of AcPhe-tRNA. It is concluded that hygromycin A is a specific inhibitor of the peptide bond formation step of protein synthesis. The action of hygromycin A on peptidyl transfer is similar to that of chloramphenicol, an antibiotic that shares some common structural features with hygromycin A. Both antibiotics inhibit the binding of C-A-C-C-A-Leu to the acceptor site of peptidyl transferase and stimulate that of C-A-C-C-A-LeuAc to the donor site of the enzyme. Moreover, hygromycin A blocks the binding of chloramphenicol to ribosomes, indicating that the binding sites of the antibiotics may be closely related. Hygromycin A is a more potent agent than chloramphenicol and binds quite strongly to ribosomes.     

Inhibition of the peptidyl transferase A-site function by 2'-O-aminoacyloligonucleotides

New “non-isomerizable” analogs of the 3′-terminus of AA-tRNA, C-A(2′Phe)H, C-A(2′Phe)Me, C-A(2′H)Phe and C-A(2′Me)Phe, were tested as acceptor substrates of ribosomal peptidyl transferase and inhibitors of the peptidyl transferase A-site function. The 3′-O-AA-derivatives were active acceptors of Ac-Phe in the peptidyl transferase reaction, while the 2′-O-AA-derivatives were completely inactive. Both 2′- and 3′-O-AA-derivatives were potent inhibitors of peptidyl transferase catalyzed Ac-Phe transfer to puromycin. The results indicate that although peptidyl transferase exclusively utilizes 3′-O-esters of tRNA as acceptor substrates, its A-site can also recognize the 3′-terminus of 2′-O-AA-tRNA.     

Investigation of the ribosomal peptidyl transferase center using a photoaffinity label

The synthesis of a peptidyl-tRNA photoaffinity analog, 2-nitro-4-azidophenoxy-4′-phenylacetyl-phenylalanyl-tRNA^Phe is described. Covalent attachment of this analog to Escherichia coli 70 S ribosomes requires poly(U)-stimulated binding prior to photolysis. Peptidyl site binding is indicated by the ability of puromycin to release the peptidyl moiety from non-photolyzed samples. Covalently attached 2-nitro-4-azidophenoxy-4-phenylacetyl-Phe-tRNA^Phe can subsequently participate in peptidyl transfer with [³H]Phe-tRNA^Phe bound at the aminoacyl site. This means that the covalent reaction does not produce sufficient distortion of the peptidyl site and its bound tRNA to inactivate the peptidyl transference. If peptidyl transfer with [³H]Phe-tRNA^Phe is allowed to proceed before photolysis, covalent reaction can still occur. In all cases, the main reaction products are two 50 S ribosomal proteins, L11 and L18. The results strongly indicate that these two proteins either form part of the peptidyl transferase center or are located adjacent to it. Presumably, α-halocarbonyl affinity reagents used previously failed to identify these two proteins because they lack easily accessible, reactive nucleophilic groups.     

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).     

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.     

Cooperative binding of 3'-fragments of transfer ribonucleic acid to the peptidyltransferase center of Escherichia coli ribosomes.

The binding of substrates to the A-site half (A′) and the P-site half (P′) of the peptidyltransferase center was measured by means of equilibrium dialysis. The tRNA fragments C-A-C-C-A-Leu and C-A-C-C-A-(N-acetyl)Leu were used as A′-site and P′-site substrates, respectively. The A′- and P′-substrates bound well to 50 S in contrast to 30 S subunits; significant binding to 23 S and 16 S RNA was also found. The binding of the P′-site substrate to 23 S RNA and 50 S subunits was very similar at various Mg²⁺ and K⁺ concentrations, indicating that the 23 S RNA is probably directly involved in the binding of the 3′-end of the peptidyl-tRNA. Cooperative effects at the peptidyltransferase center were found using chloramphenicol and deacylated tRNA as competitors, which completely inhibited the substrate binding to one site whilst drastically stimulating binding to the other. Chloramphenicol inhibited the binding of the A′-site substrate C-A-C-C-A-Leu, whereas the binding of the corresponding P′-site substrate was stimulated. In contrast, deacylated tRNA blocked the binding of the P′-site substrate, but stimulated the corresponding A′-site binding. Similarly, the trinucleotide C_p,C_pA inhibited binding of the P′-site substrate (showing complete inhibition at 70 μm) whereas binding of the A′-site substrate was slightly stimulated at concentrations below 70 μm.     

An intact ribose moiety at A2602 of 23S rRNA is key to trigger peptidyl-tRNA hydrolysis during translation termination

Peptide bond formation and peptidyl-tRNA hydrolysis are the two elementary chemical reactions of protein synthesis catalyzed by the ribosomal peptidyl transferase ribozyme. Due to the combined effort of structural and biochemical studies, details of the peptidyl transfer reaction have become increasingly clearer. However, significantly less is known about the molecular events that lead to peptidyl-tRNA hydrolysis at the termination phase of translation. Here we have applied a recently introduced experimental system, which allows the ribosomal peptidyl transferase center (PTC) to be chemically engineered by the introduction of non-natural nucleoside analogs. By this approach single functional group modifications are incorporated, thus allowing their functional contributions in the PTC to be unravelled with improved precision. We show that an intact ribose sugar at the 23S rRNA residue A2602 is crucial for efficient peptidyl-tRNA hydrolysis, while having no apparent functional relevance for transpeptidation. Despite the fact that all investigated active site residues are universally conserved, the removal of the complete nucleobase or the ribose 2′-hydroxyl at A2602, U2585, U2506, A2451 or C2063 has no or only marginal inhibitory effects on the overall rate of peptidyl-tRNA hydrolysis. These findings underscore the exceptional functional importance of the ribose moiety at A2602 for triggering peptide release.     

Puromycin reaction of the A-site bound peptidyl-tRNA.

AcPhe2-tRNA(Phe) which appears in ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is able to react quantitatively with puromycin in the absence of EF-G. One could readily explain this fact to be the consequence of spontaneous translocation. However, a detailed study of kinetics of puromycin reaction carried out with the use of viomycin (inhibitor of translocation) and the P-site test revealed that, apart from spontaneous translocation, this peptidyl-tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction triggers conformational changes in the A-site ribosomal complex bringing the 3'-end of a newly synthesized peptidyl-tRNA nearer to the peptidyl site of peptidyltransferase center. This is detected functionally as a highly pronounced ability of such a peptidyl-tRNA to react with puromycin.     

Dual actions of viomycin on the ribosomal functions.

Viomycin was observed to inhibit poly[U]- or f2 RNA-directed protein synthesis in an $\text{E. coli}$ cell-free system. The former was more profoundly affected than the latter. Both initiation complex formation on the 30S ribosomal subunit and on 70S ribosomes were prevented by the antibiotic. In the peptide chain elongation process, viomycin did not significantly affect aminoacyl-tRNA binding to ribosomes and the peptidyl transferase reaction, but markedly inhibit translocation of peptidyl-tRNA from the acceptor site to the donor site. The mechanism of action of the drug appeared to be unique.     

The 2'-OH group of the peptidyl-tRNA stabilizes an active conformation of the ribosomal PTC

The ribosome accelerates the rate of peptidyl transfer by >10(6)-fold relative to the background rate. A widely accepted model for this rate enhancement invokes entropic effects whereby the ribosome and the 2'-OH of the peptidyl-tRNA substrate precisely position the reactive moieties through an extensive network of hydrogen bonds that allows proton movement through them. Some studies, however, have called this model into question because they find the 2'-OH of the peptidyl-tRNA to be dispensable for catalysis. Here, we use an in vitro reconstituted translation system to resolve these discrepancies. We find that catalysis is at least 100-fold slower with the dA76-substituted peptidyl-tRNA substrate and that the peptidyl transferase centre undergoes a slow inactivation when the peptidyl-tRNA lacks the 2'-OH group. Additionally, the 2'-OH group was found to be critical for EFTu binding and peptide release. These findings reconcile the conflict in the literature, and support a model where interactions between active site residues and the 2'-OH of A76 of the peptidyl-tRNA are pivotal in orienting substrates in this active site for optimal catalysis.     

Modulation of the rate of peptidyl transfer on the ribosome by the nature of substrates

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.     

The Identification of the Determinants of the Cyclic, Sequential Binding of Elongation Factors Tu and G to the Ribosome

Experiments dedicated to gaining an understanding of the mechanism underlying the orderly, sequential association of elongation factor Tu (EF-Tu) and elongation factor G (EF-G) with the ribosome during protein synthesis were undertaken. The binding of one EF is always followed by the binding of the other, despite the two sharing the same—or a largely overlapping—site and despite the two having isosteric structures. Aminoacyl-tRNA, peptidyl-tRNA, and deacylated-tRNA were bound in various combinations to the A-site, P-site, or E-site of ribosomes, and their effect on conformation in the peptidyl transferase center, the GTPase-associated center, and the sarcin/ricin domain (SRD) was determined. In addition, the effect of the ribosome complexes on sensitivity to the ribotoxins sarcin and pokeweed antiviral protein and on the binding of EF-G•GTP were assessed. The results support the following conclusions: the EF-Tu ternary complex binds to the A-site whenever it is vacant and the P-site has peptidyl-tRNA; and association of the EF-Tu ternary complex is prevented, simply by steric hindrance, when the A-site is occupied by peptidyl-tRNA. On the other hand, the affinity of the ribosome for EF-G•GTP is increased when peptidyl-tRNA is in the A-site, and the increase is the result of a conformational change in the SRD. We propose that peptidyl-tRNA in the A-site is an effector that initiates a series of changes in tertiary interactions between nucleotides in the peptidyl transferase center, the SRD, and the GTPase-associated center of 23S rRNA; and that the signal, transmitted through a transduction pathway, informs the ribosome of the position of peptidyl-tRNA and leads to a conformational change in the SRD that favors binding of EF-G.     

Identification of proteins at the peptidyl-tRNA binding site of rat liver ribosomes

Summary We have identified proteins involved in the peptidyl-tRNA-binding site of rat liver ribosomes, using an affinity label designed specifically to probe the P-site in eukaryotic peptidyl transferase. The label is a 3-terminal pentanucleotide fragment of N-acetylleucyl-tRNA in which mercury atoms have been added at the C-5 position of the three cytosine residues. This mercurated fragment can bind to rat liver peptidyl transferase and function as a donor of N-acetylleucine to puromycin. Concommitant with this binding, the mercury atoms present in the fragment can form a covalent linkage with a small number of ribosomal proteins. The major proteins labeled by this reagent are L5 and L36A. Four protein spots are found labeled to a lesser extent: L10, L7/7a, L3/4 and L25/31. Each of these proteins, therefore, is implicated in the binding of the 3-terminus of peptidyl-tRNA.The results presented here are correlated with other investigations of the structure-function aspects of rat liver peptidyl transferase. Using these data, we have constructed a model for the arrangement of proteins within this active site.     

The peptidyltransferase center of Escherichia coli ribosomes: Binding sites for the cytidine 3′-phosphate residues of the aminoacyl-tRNA 3′-terminus and the interrelationships between the acceptor and donor sites

The substrate specificity of the acceptor site of peptidyltransferase of Escherichia coli 70 S ribosomes was investigated in Ac-Phe-tRNA·poly(U)·70 S ribosome (system A) and tRNA^Phe·poly(U)·C-A-C-C-A-Phe·70 S ribosome (system B) systems by using C-C-A-Gly, C-C-A-Phe, C-A-Gly and C-A-Phe as analogs of the 3′-terminus of aminoacyl-tRNA. It was found that an addition of C_p residue to C-A-Gly and C-A-Phe resulted in an increase of the acceptor activity in system A; the increase is more remarkable for C-A-Gly than for C-A-Phe, while the acceptor activities of C-C-A-Gly and C-C-A-Phe are roughly similar. On the other hand, dramatically increased binding affinities of C-C-A-Phe and C-C-A-Gly relative to C-A-Phe and C-A-Gly for the A site of peptidyltransferase were observed in system B using an inhibition assay; C-C-A-Phe binds much more strongly than C-C-A-Gly. The results indicate the important role of the third C_p residue and the aminoacyl moiety of the 3′-terminus of aminoacyl-tRNA in the interaction with the acceptor site of peptidyltransferase, as well as the existence of cooperative effects between A and P sites of peptidyltransferase. These effects, depending on an occupancy of P site, may significantly influence the specificity of the peptidyltransferase A site.