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.     

Protein L16 of the Escherichia coli ribosome: possible role in protein biosynthesis

We show that Escherichia coli 50S ribosomal subunits depleted of protein L16 can nevertheless catalyze the transfer of the peptide moiety from fMet-tRNA to puromycin, being, however, unable to use a fragment CACCA-Phe as an acceptor substrate. On the other hand, we found that protein L16 as well as its large fragment (amino acids 10-136) both interact with tRNA in solution (Kd approximately 10(-7) M). Moreover, L16 interacts with CACCA-Phe in solution as well as protects 3' end of tRNA from the enzymatic degradation. We suggest that L16, although not being the peptidyl transferase as such, is involved in the binding of the 3' end cytidines of tRNA into the ribosomal A site.     

Role of the ribosomal protein L27 revealed by single‐molecule FRET study

The ribosome is a ribozyme. However, in bacterial ribosomes, the N‐terminus of L27 is located within the peptidyl transfer center. The roles of this protein in real time remain unclear. We present single‐molecule fluorescence resonance energy transfer (FRET) studies of tRNA dynamics at the peptidyl transfer center in ribosomes containing either wild type (WT) L27, or L27 mutants with A2H3, A2H3K4 or nine N‐terminal residues removed. Removing L27's first three N‐terminal residues or mutating a single residue, K4, reduces the formation of a stable peptidyl tRNA after translocation. These results imply that L27 stabilizes the peptidyl tRNA and residue K4 contributes significantly to the stabilization.     

Role of ribosomal protein L27 in peptidyl transfer

The current view of ribosomal peptidyl transfer is that the ribosome is a ribozyme and that ribosomal proteins are not involved in catalysis of the chemical reaction. This view is largely based on the first crystal structures of bacterial large ribosomal subunits that did not show any protein components near the peptidyl transferase center (PTC). Recent crystallographic data on the full 70S ribosome from Thermus thermophilus, however, show that ribosomal protein L27 extends with its N-terminus into the PTC in accordance with independent biochemical data, thus raising the question of whether the ribozyme picture is strictly valid. We have carried out extensive computer simulations of the peptidyl transfer reaction in the T. thermophilus ribosome to address the role of L27. The results show a reaction rate similar to that obtained in earlier simulations of the Haloarcula marismortui reaction. Furthermore, deletion of L27 is predicted to only give a minor rate reduction, in agreement with biochemical data, suggesting that the ribozyme view is indeed valid. The N-terminus of L27 is predicted to interact with the A76 phosphate group of the A-site tRNA, thereby explaining the observed impairment of A-site substrate binding for ribosomes lacking L27. Simulations are also reported for the reaction with puromycin, an A-site tRNA analogue which lacks the A76 phosphate group. The calculated energetics shows that this substrate can cause a downward p K a shift of L27 and that the reaction proceeds faster with the L27 N-terminus deprotonated, in contrast to the situation with aminoacyl-tRNA substrates. These results could explain the observed differences in pH dependence between the puromycin and C-puromycin reactions, where the former reaction has been seen to depend on an additional ionizing group besides the attacking amine, and our model predicts this ionizing group to be the N-terminal amine of L27.     

The properties of the complex between ribosomal protein L2 and tRNA

Escherichia coli ribosomal protein L2 interacts with fMet-tRNA^Met and NacPhe-tRNA^Phe in solution, protecting their 3'-ends from enzymatic degradation. At the same time L2 enhances the rate of spontaneous hydrolysis of the ester bonds between terminal riboses and amino acyl moieties of these two peptidyl-tRNA analogues. L2 has, however, only a slight effect on the rate of spontaneous deacylation of aminoacyltRNAs. We suggest that the role of L2 is in the fixation of the aminoacyl stem of tRNA to the ribosome at its P-site, and speculate that this protein is directly involved in the peptidyl transferase (PT) reaction. Peptidyl transferase Protein L2 tRNA-protein complex     

Transit of tRNA through the Escherichia coli ribosome: cross-linking of the 3' end of tRNA to ribosomal proteins at the P and E sites

Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at their 3' termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome. When bound to the P site of poly(U)-programmed ribosomes, deacylated tRNA(Phe), Phe-tRNA(Phe) and N-acetyl-Phe-tRNA(Phe) probes labeled protein L27 and two main sites within domain V of the 23S RNA. In contrast, deacylated tRNA(Phe) bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA. In the absence of poly(U), the deacylated tRNA(Phe) probe also labeled protein L1. Cross-linking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27. In the course of this process, tRNA passes through the intermediate P/E binding state. These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order. Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker. The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S 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)     

The human large subunit ribosomal protein L36A-like contacts the CCA end of P-site bound tRNA

Periodate-oxidized tRNA (tRNAox), the 2′,3′-dialdehyde derivative of tRNA, was used as a zero-length active site-directed affinity labeling reagent, to covalently label proteins at the binding site for the 3′-end of tRNA on human 80S ribosomes. When human 80S ribosomes were reacted with tRNA^Aspox positioned at the P-site, in the presence of an appropriate 12 mer mRNA, a set of two tRNAox-labeled ribosomal proteins (rPs) was observed. The majorily labeled protein was identified as the large subunit rP L36a-like (RPL36AL) by means of mass spectrometry. Intact tRNA^Asp competed with tRNA^Aspox for the binding to the P-site, by preventing tRNA-protein cross-linking with RPL36AL. Altogether, the data presented in this report are consistent with the presence of RPL36AL at or near the binding site for the CCA end of the tRNA substrate positioned at the P-site of human 80S ribosomes. It is the first time that a ribosomal protein is found in an intimate contact (i.e. at a zero-distance) with a nucleotide of the conserved CCA terminus of P-site tRNA which is the substrate of peptidyl transferase reaction. RPL36AL which is strongly conserved in eukaryotes belongs to the L44e family of rPs, a representative of which is Haloarcula marismortui RPL44e.     

The mechanism of covalent reaction of bromoacetyl-phenylalanyl-transfer RNA with the peptidyl-transfer RNA binding site of the Escherichia coli ribosome

Results are presented to prove that bromoacetyl-phenylalanyl-transfer RNA reacts covalently with 50 S ribosomal proteins L2 and L27 while it is bound correctly to the peptidyl site on the 70 S ribosome. Attachment of the BrAcPhe moiety to tRNA causes a 100-fold enhancement of its reactivity with ribosomes. This reactivity closely parallels binding of tRNA whether measured by poly(U) stimulation or competition with deacylated tRNA. BrAcPhe-tRNA can bind correctly to the P site as judged by puromycin releasibility and lack of tetracycline inhibition. Little significant reaction of BrAcPhe-tRNA with L2 and L27 occurs during procedures used to purify and analyze ribosomal proteins. If ribosomes are first incubated with BrAcPhe-tRNA and subsequently treated with puromycin before analysis, little inhibition of the covalent reaction with L2 and L27 is observed. In contrast, a few minor reaction products are markedly suppressed. Covalently attached BrAcPhe-tRNA is still capable of accepting an amino acid from Phe-tRNA or puromycin. The products from this reaction are found attached to proteins L2 and L27 and to a lesser extent to L15 and L16. This shows that true affinity labeling of proteins in the peptidyl binding site has been accomplished.Some covalent reaction of BrAcPhe-tRNA with the 30 S protein S18 is also observed. This reaction is not poly(U)-dependent, however, and S18-reacted BrAcPhe-tRNA is not capable of peptide bond formation with Phe-tRNA. It seems likely that reaction with S18 results from a non-functional interaction of the affinity label with the ribosome.     

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.     

Accommodating the bacterial decoding release factor as an alien protein among the RNAs at the active site of the ribosome

The decoding release factor （RF） triggers termination of protein synthesis by functionally mimicking a tRNA to span the decoding centre and the peptidyl transferase centre （PTC） of the ribosome. Structurally, it must fit into a site crafted for a tRNA and surrounded by five other RNAs, namely the adjacent peptidyl tRNA carrying the completed polypeptide, the mRNA and the three rRNAs. This is achieved by extending a structural domain from the body of the protein that results in a critical conformational change allowing it to contact the PTC. A structural model of the bacterial termination complex with the accommodated RF shows that it makes close contact with the first, second and third bases of the stop codon in the mRNA with two separate loops of structure＂ the anticodon loop and the loop at the tip of helix orS. The anticodon loop also makes contact with the base following the stop codon that is known to strongly influence termination efficiency. It confirms the close contact of domain 3 of the protein with the key RNA structures of the PTC. The mRNA signal for termination includes sequences upstream as well as downstream of the stop codon, and this may reflect structural restrictions for specific combinations of tRNA and RF to be bound onto the ribosome together. An unbiased SELEX approach has been investigated as a tool to identify potential rRNA-binding contacts of the bacterial RF in its different binding conformations within the active centre of the ribosome.     

Reconstitution of Escherichia coli 50S ribosomal subunits containing puromycin-modified L23: functional consequences

In previous work we have shown that both puromycin [Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274] and p-azidopuromycin [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Coooperman, B. S. (1982) Biochemistry 21, 3809-3817] site specifically photoaffinity label protein L23 to the highest extent of any Escherichia coli ribosomal protein. In this work we demonstrate that L23 that has been photoaffinity labeled within a 70S ribosome by puromycin (puromycin-L23) can be separated from unmodified L23 by reverse-phase high-performance liquid chromatography (RP-HPLC) and further that puromycin-L23 can reconstitute into 50S subunits when added in place of unmodified L23 to a reconstitution mixture containing the other 50S components in unmodified form. We have achieved a maximum incorporation of 0.5 puromycin-L23 per reconstituted 50S subunit. As compared with reconstituted 50S subunits either containing unmodified L23 or lacking L23, reconstituted 50S subunits containing 0.4-0.5 puromycin-L23 retain virtually all (albeit low) peptidyl transferase activity but only 50-60% of mRNA-dependent tRNA binding stimulation activity. We conclude that although L23 is not directly at the peptidyl transferase center, it is sufficiently close that puromycin-L23 can interfere with tRNA binding. This conclusion is consistent with a number of other experiments placing L23 close to the peptidyl transferase center but is difficult to reconcile with immunoelectron microscopy results placing L23 near the base of the 50S subunit on the side facing away from the 30S subunit [Hackl, W., & St?ffler-Meilicke, M. (1988) Eur. J. Biochem. 174, 431-435].     

New photoreactive tRNA derivatives for probing the peptidyl transferase center of the ribosome

Three new photoreactive tRNA derivatives have been synthesized for use as probes of the peptidyl transferase center of the ribosome. In two of these derivatives, the 3' adenosine of yeast tRNA(Phe) has been replaced by either 2-azidodeoxyadenosine or 2-azido-2'-O-methyl adenosine, while in a third the 3'-terminal 2-azidodeoxyadenosine of the tRNA is joined to puromycin via a phosphoramidate linkage to generate a photoreactive transition-state analog. All three derivatives bind to the P site of 70S ribosomes with affinities similar to that of unmodified tRNA(Phe) and can be cross-linked to components of the 50S ribosomal subunit by irradiation with near-UV light. Characteristic differences in the cross-linking patterns suggest that these tRNA derivatives can be used to follow subtle changes in the position of the tRNA relative to the components of the peptidyl transferase center.     

Localization of ribosomal protein L27 at the peptidyl transferase centre of the 50 S subunit, as determined by immuno-electron microscopy

Summary Protein L27 has been localized on the ribosomal surface by immuno-electron microscopy by using antibodies specific for Escherichia coli L27, and by reconstituting 50 S subunits from an E. coli mutant, which lacks protein L27, with the homologous protein from Bacillus subtilis and using antibodies specific for the B. subtilis protein. With both approaches, protein L27 has been located at the base of the central protuberance at the interface side of the 50 S particle and thus in proximity to the peptidyl transferase centre. The immuno-electron microscopic data also suggest that the interface region of the 50 S particle is not as flat as most of the proposed three-dimensional models suggest, but instead there is a significant depression.     

Mutations in domain V of the 23S ribosomal RNA of Bacillus subtilis that inactivate its protein folding property in vitro

The active site of a protein folding reaction is in domain V of the 23S rRNA in the bacterial ribosome and its homologs in other organisms. This domain has long been known as the peptidyl transferase center. Domain V of Bacillus subtilis is split into two segments, the more conserved large peptidyl transferase loop (RNA1) and the rest (RNA2). These two segments together act as a protein folding modulator as well as the complete domain V RNA. A number of site-directed mutations were introduced in RNA1 and RNA2 of B.subtilis, taking clues from reports of these sites being involved in various steps of protein synthesis. For example, sites like G2505, U2506, U2584 and U2585 in Escherichia coli RNA1 region are protected by deacylated tRNA at high Mg²⁺ concentration and A2602 is protected by amino acyl tRNA when the P site remains occupied already. Mutations A2058G and A2059G in the RNA1 region render the ribosome Ery^r in E.coli and Lnc^r in tobacco chloroplast. Sites in P loop G2252 and G2253 in E.coli are protected against modification by the CCA end of the P site bound tRNA. Mutations were introduced in corresponding nucleotides in B.subtilis RNA1 and RNA2 of domain V. The mutants were tested for refolding using unfolded protein binding assays with unfolded carbonic anhydrase. In the protein folding assay, the mutants showed partial to complete loss of this activity. In the filter binding assay for the RNA–refolding protein complex, the mutants showed an extent of protein binding that agreed well with their protein folding activity.     

Activities of the peptidyl transferase center of ribosomes lacking protein L27

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.     

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.     

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.