The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop.

A photo-reactive diazirine derivative was attached to the 2-thiocytidine residue at position 32 of tRNA(Arg)I from Escherichia coli. This modified tRNA was bound under suitable conditions to the A, P or E site of E.coli ribosomes. After photo-activation of the diazirine label, the sites of cross-linking to 16S rRNA were identified by our standard procedures. Each of the three tRNA binding sites showed a characteristic pattern of cross-linking. From tRNA at the A site, a major cross-link was observed to position 1378 of the 16S RNA, and a minor one to position 936. From the P site, there were major cross-links to positions 693 and to 957 and/or 966, as well as a minor cross-link to position 1338. The E site bound tRNA showed major cross-links to position 693 (identical to that from the P site) and to positions 1376/1378 (similar, but not identical, to the cross-link observed from the A site). Immunological analysis of the concomitantly cross-linked ribosomal proteins indicated that S7 was the major target of cross-linking from all three tRNA sites, with S11 as a minor product. The results are discussed in terms of the overall topography of the decoding region of the 30S ribosomal subunit.     

Topography of the E site on the Escherichia coli ribosome.

Three photoreactive tRNA probes have been utilized in order to identify ribosomal components that are in contact with the aminoacyl acceptor end and the anticodon loop of tRNA bound to the E site of Escherichia coli ribosomes. Two of the probes were derivatives of E. coli tRNA(Phe) in which adenosines at positions 73 and 76 were replaced by 2-azidoadenosine. The third probe was derived from yeast tRNA(Phe) by substituting wyosine at position 37 with 2-azidoadenosine. Despite the modifications, all of the photoreactive tRNA species were able to bind to the E site of E. coli ribosomes programmed with poly(A) and, upon irradiation, formed covalent adducts with the ribosomal subunits. The tRNA(Phe) probes modified at or near the 3' terminus exclusively labeled protein L33 in the 50S subunit. The tRNA(Phe) derivative containing 2-azidoadenosine within the anticodon loop became cross-linked to protein S11 as well as to a segment of the 16S rRNA encompassing the 3'-terminal 30 nucleotides. We have located the two extremities of the E site-bound tRNA on the ribosomal subunits according to the positions of L33, S11 and the 3' end of 16S rRNA defined by immune electron microscopy. Our results demonstrate conclusively that the E site is topographically distinct from either the P site or the A site, and that it is located alongside the P site as expected for the tRNA exit site.     

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.     

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.     

Mapping the rRNA neighborhood of the acceptor end of tRNA in the ribosome.

In order to map the rRNA environment of the acceptor end of tRNA in th e ribosome, hydroxyl radicals were generated in situ from Fe(II) attached via an EDTA linker to the 5' end of tRNA. Nucleotides in rRNA cleaved by the radicals were identified by primer extension, and assigned to the ribosomal A, P and E sites by standard criteria. In the A site, cleavages were found in the 2555-2573 region of 23S rRNA, around bases previously shown to be protected by A site tRNA, and in the alpha-sarcin loop, the site of interaction of elongation factors EF-Tu and EF-G. P site cleavages occurred in the 2250 loop, where a base pair is made with C74 of tRNA; and around the 2493 region in domain V. Interestingly, two clusters of nucleotides in 23S rRNA are accessible to both A site and P site tRNA probes. The first cluster is in the 1940-1965 region of domain IV, around the site of affinity labeling by the 3' end of tRNA, and the second cluster is around the bulged adenosine A2602, whose accessibility to chemical probes is enhanced by P site tRNA and decreased by A site tRNA. From the E site, cleavages occur in the 2390-2440 region, surrounding C2394, a base protected from dimethyl sulfate by E site tRNA, and in the phylogenetically variable stem at positions 1860/1880 of domain IV. Unexpectedly, no cleavages were detected in the central loop of domain V of 23S rRNA.     

Structures of deacylated tRNA mimics bound to the E site of the large ribosomal subunit

During translation, tRNAs cycle through three binding sites on the ribosome: the A, the P, and the E sites. We have determined the structures of complexes between the Haloarcula marismortui large ribosomal subunit and two different E site substrates: a deacylated tRNA acceptor stem minihelix and a CCA-acceptor end. Both of these tRNA mimics contain analogs of adenosine 76, the component responsible for a large proportion of E site binding affinity. They bind in the center of the loop-extension of protein L44e, and make specific contacts with both L44e and 23S rRNA including bases that are conserved in all three kingdoms of life. These contacts are consistent with the footprinting, protection, and cross-linking data that have identified the E site biochemically. These structures explain the specificity of the E site for deacylated tRNAs, as it is too small to accommodate any relevant aminoacyl-tRNA. The orientation of the minihelix suggests that it may mimic the P/E hybrid state. It appears that the E site on the 50S subunit was formed by only RNA in the last common ancestor of the three kingdoms, since the proteins at the E sites of H. marismortui and Deinucoccus radiodurans large subunits are not homologous.     

The side-by-side model of two tRNA molecules allowing the alpha-helical conformation of the nascent polypeptide during the ribosomal transpeptidation.

Lim and Spirin [25] proposed a preferable conformation of the nascent peptide during the ribosomal transpeptidation. Spirin and Lim [26] excluded the possibilities of the side-by-side model proposed by Johnson et al [13] and the three-tRNA binding model (A, P and E sites) of Rheinberger and Nierhaus [3]. However, a slight conformational change at the 3' end regions of both A and P site tRNA molecules can enable the three different tRNA binding models to converge. With a modification of the angles of the ribose rings of both anticodon and mRNA this model can also be related to the model of Sundaralingam et al [19]. In this model of E coli rRNA the 3' end sequence ACCA76 or GCCA76 of P site tRNA is base-paired to UGGU810 of 23S rRNA, while the ACC75 or GCC75 of A site tRNA are base-paired to GGU1621 23S rRNA. The conformation of the A76 of A site tRNA is necessarily different from that of P site tRNA, at least during the course of the transpeptidation. The A76 of A site tRNA overlaps the binding region of puromycin. The C1400 of 16S rRNA in this model is located at a distance of 4 A from the 5' end of the anticodon of P site tRNA [14] and 17 A from the 5' end of the anticodon of A site tRNA [15]. It is also shown that a considerable but reasonable modification in the conformation of the anticodon loops could lead to accommodation of three deacylated tRNA(Phe) molecules at a time on 70S ribosome in the presence of poly(U) as observed experimentally [6]. A sterochemical explanation for the negatively-linked allosteric interactions between the A and E sites is also shown in the present model.     

Interactions between 23S rRNA and tRNA in the ribosomal E site

Interactions between tRNA or its analogs and 23S rRNA in the large ribosomal subunit were analyzed by RNA footprinting and by modification-interference selection. In the E site, tRNA protected bases G2112, A2392, and C2394 of 23S rRNA. Truncated tRNA, lacking the anticodon stem-loop, protected A2392 and C2394, but not G2112, and tRNA derivatives with a shortened 3' end protected only G2112, but not A2392 or C2394. Modification interference revealed C2394 as the only accessible nucleotide in 23S rRNA whose modification interferes with binding of tRNA in the large ribosomal subunit E site. The results suggest a direct contact between A76 of tRNA A76 and C2394 of 23S rRNA. Protections at G2112 may reflect interaction of this 23S rRNA region with the tRNA central fold.     

The ribosomal environment of tRNA: crosslinks to rRNA from positions 8 and 20:1 in the central fold of tRNA located at the A, P, or E site.

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl) uridine ("acp3U") at position 20:1 of lupin tRNAMet was coupled to a photoreactive diazirine derivative. Similarly, the 4-thiouridine at position 8 of Escherichia coli tRNAPhe was modified with an aromatic azide. Each of the derivatized tRNAs was bound to E. coli ribosomes in the presence of suitable mRNA analogues, under conditions specific for the A, P, or E sites. After photoactivation of the diazirine or azide groups, the sites of crosslinking from the tRNAs to 16S or 23S rRNA were analyzed by our standard procedures, involving a combination of ribonuclease H digestion and primer extension analysis. The crosslinked ribosomal proteins were also identified. The results for the rRNA showed a well-defined series of crosslinks to both the 16S and 23S molecules, the most pronounced being (1) an entirely A-site-specific crosslink from tRNA position 20:1 to the loop-end region (nt 877-913) of helix 38 of the 23S RNA (a region that has not so far been associated at all with tRNA binding), and (2) a largely P-site-specific crosslink from tRNA position 8 to nt 2111-2112 of the 23S RNA (nt 2112 being a position that has previously been identified in footprinting studies as belonging to the ribosomal E site). The data are compared with results from a parallel study of crosslinks from position 47 (also in the central fold of the tRNA), as well as with previously published crosslinks from the anticodon loop (positions 32, 34, and 37) and the CCA-end region (position 76, and the aminoacyl residue).     

Binding of the 3'' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA.     

Peptidyl transferase antibiotics perturb the relative positioning of the 3'-terminal adenosine of P/P'-site-bound tRNA and 23S rRNA in the ribosome.

A range of antibiotic inhibitors that act within the peptidyl transferase center of the ribosome were examined for their capacity to perturb the relative positioning of the 3' end of P/P'-site-bound tRNA and the Escherichia coli ribosome. The 3'-terminal adenosines of deacylated tRNA and N-Ac-Phe-tRNA were derivatized at the 2 position with an azido group and the tRNAs were cross-linked to the ribosome on irradiation with ultraviolet light at 365 nm. The cross-links were localized on the rRNA within extended versions of three previously characterized 23S rRNA fragments F1', F2', and F4' at nucleotides C2601/A2602, U2584/U2585 (F1'), U2506 (F2'), and A2062/C2063 (F4'). Each of these nucleotides lies within the peptidyl transferase loop region of the 23S rRNA. Cross-links were also formed with ribosomal proteins L27 (strong) and L33 (weak), as shown earlier. The antibiotics sparsomycin, chloramphenicol, the streptogramins pristinamycin IA and IIA, gougerotin, lincomycin, and spiramycin were tested for their capacity to alter the identities or yields of each of the cross-links. Although no new cross-links were detected, each of the drugs produced major changes in cross-linking yields, mainly decreases, at one or more rRNA sites but, with the exception of chloramphenicol, did not affect cross-linking to the ribosomal proteins. Moreover, the effects were closely similar for both deacylated and N-Ac-Phe-tRNAs, indicating that the drugs selectively perturb the 3' terminus of the tRNA. The strongest decreases in the rRNA cross-links were observed with pristinamycin IIA and chloramphenicol, which correlates with their both producing complex chemical footprints on 23S rRNA within E. coli ribosomes. Furthermore, gougerotin and pristinamycin IA strongly increased the yields of fragments F2' (U2506) and F4' (U2062/C2063), respectively. The results obtained with an RNAse H approach correlate well with primer extension data implying that cross-linking occurs primarily to the bases. Both sets of data are also consistent with the results of earlier rRNA footprinting experiments on antibiotic-ribosome complexes. It is concluded that the antibiotics perturb the relative positioning of the 3' end of the P/P'-site-bound tRNA and the peptidyl transferase loop region of 23S rRNA.     

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.     

Neighbourhood of the central fold of the tRNA molecule bound to the E. coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop.

The neighbourhood of the dihydrouridine loop of tRNA molecule bound to E. coli ribosome has been studied by affinity labeling, using modified tRNAs carrying photoreactive azidonitrophenyl probes attached to the 3-(3-amino-3-carboxypropyl)-uridine located at position 20:1 of Lupin methionine elongator tRNA. The maximum distance between the pyrimidine ring and the azido group estimated for the two probes employed in this study is 10-11 A and 18-19 A, respectively. Cross-linking of the uncharged, modified tRNAs has been studied with poly(A, U, G) as a message, under conditions directing uncharged tRNAs preferentially to the ribosomal P-site. Modified tRNAs bind covalently to both ribosomal subunits with high yields upon irradiation of the respective non-covalent complexes. Proteins S7, L33 and L1 have been consistently found cross-linked to tRNAs modified with both probes, and S5 and L5 to tRNA modified with the longer probe. Surprisingly, an S5-tRNA cross-linking product is reproducibly found in a protein fraction prepared from the purified 50S subunit. Cross-linking to rRNAs is significant only for the longer probe and is stimulated 2-4 fold in the presence of poly(A,U,G). The cross-linking sites are located between nucleotides 1302 and 1398 in 16S rRNA and between nucleotides 2281 and 2358 in 23S rRNA.     

Selective isolation and detailed analysis of intra-RNA cross-links induced in the large ribosomal subunit of E. coli: a model for the tertiary structure of the tRNA binding domain in 23S RNA.

Intramolecular RNA cross-links were induced within the large ribosomal subunit of E. coli by mild ultraviolet irradiation. Regions of the 23S RNA previously implicated in interactions with ribosomal-bound tRNA were then specifically excised by addressed cleavage using ribonuclease H, in conjunction with synthetic complementary decadeoxyribonucleotides. Individual cross-linked fragments within these regions released by such 'directed digests' were isolated by two-dimensional gel electrophoresis and the sites involved in the cross-links determined using classical oligonucleotide analysis techniques. Using this approach, seven 'new' cross-links could be precisely localised, between positions 1782 and 2608-2609, 1940 and 2554, 1941-1942 and 1964-1965, 1955 and 2552-2553, 2145-2146 and 2202, 2518-2519 and 2544-2545, and between positions 2790-2791 and 2892-2895 in the 23S RNA sequence. These data, in conjunction with data from RNA-protein cross-linking studies carried out in our laboratory, were used to define a model for the tertiary organisation of the tRNA binding domain of 23S RNA 'in situ', in which the specific nucleotides associated with tRNA binding in the 'A' and 'P' sites are clustered at the base of the 'central protuberance' of the 50S subunit.     

Positioning of CCA-arms of the A- and the P-tRNAs towards the 28S rRNA in the human ribosome

Nucleotides of 28S rRNA involved in binding of the human 80S ribosome with acceptor ends of the A site and the P site tRNAs were determined using two complementary approaches, namely, cross-linking with application of tRNA^Asp analogues substituted with 4-thiouridine in position 75 or 76 and hydroxyl radical footprinting with the use of the full sized tRNA and the tRNA deprived of the 3′-terminal trinucleotide CCA. In general, these 28S rRNA nucleotides are located in ribosomal regions homologous to the A, P and E sites of the prokaryotic 50S subunit. However, none of the approaches used discovered interactions of the apex of the large rRNA helix 80 with the acceptor end of the P site tRNA typical with prokaryotic ribosomes. Application of the results obtained to available atomic models of 50S and 60S subunits led us to a conclusion that the A site tRNA is actually present in both A/A and A/P states and the P site tRNA in the P/P and P/E states. Thus, the present study gives a biochemical confirmation of the data on the structure and dynamics of the mammalian ribosomal pretranslocation complex obtained with application of cryo-electron microscopy and single-molecule FRET [Budkevich et al., 2011]. Moreover, in our study, particular sets of 28S rRNA nucleotides involved in oscillations of tRNAs CCA-termini between their alternative locations in the mammalian 80S ribosome are revealed.     

Interaction of unfolded tRNA with the 3''-terminal region of E. coli 16S ribosomal RNA.

Fragments of tRNA possessing a free TpsiC-loop or a free D-loop form stable complexes with the colicin fragment (1494-1542) of 16S ribosomal RNA from E. coli. The colicin fragment does not bind to tRNA in which the T-loop and the D-loop are involved in tertiary interactions. Colicin cleavage of the 16S rRNA from E. coli is inhibited by aminoacyl-tRNA or tRNA fragments, indicating that a similar interaction may take place on the intact 70S ribosomes. The oligonucleotide d(G-T-T-C-G-A)homologous to the conserved sequence G-T-psi-C-Pu-(m1)A in the TpsiC-region of many elongator tRNAs binds to the conserved sequence U-C-G-mU-A-A-C (1495-1501) of the 16S rRNA. It is suggested that the 3'-end of the 16S rRNA may provide the part of the binding site for the elongator tRNAs on bacterial ribosomes.     

Contribution of ribosomal residues to P-site tRNA binding

Structural studies have revealed multiple contacts between the ribosomal P site and tRNA, but how these contacts contribute to P-tRNA binding remains unclear. In this study, the effects of ribosomal mutations on the dissociation rate (k_off) of various tRNAs from the P site were measured. Mutation of the 30S P site destabilized tRNAs to various degrees, depending on the mutation and the species of tRNA. These data support the idea that ribosome-tRNA interactions are idiosyncratically tuned to ensure stable binding of all tRNA species. Unlike deacylated elongator tRNAs, N-acetyl-aminoacyl-tRNAs and tRNA^fMet dissociated from the P site at a similar low rate, even in the presence of various P-site mutations. These data provide evidence for a stability threshold for P-tRNA binding and suggest that ribosome-tRNA^fMet interactions are uniquely tuned for tight binding. The effects of 16S rRNA mutation G1338U were suppressed by 50S E-site mutation C2394A, suggesting that G1338 is particularly important for stabilizing tRNA in the P/E site. Finally, mutation C2394A or the presence of an N-acetyl-aminoacyl group slowed the association rate (k_on) of tRNA dramatically, suggesting that deacylated tRNA binds the P site of the ribosome via the E site.     

Covalent cross-linking of tRNAGly1 to the ribosomal P site via the dihydrouridine loop

The dihydrouracil residue at position 20 of Escherichia coli tRNAGly1 has been replaced by the photoaffinity reagent, N-(4-azido-2-nitrophenyl)glycyl hydrazide (AGH). The location of the substituent was confirmed by the susceptibility of the modified tRNA to cleavage with aniline. When N-acetylglycyl-tRNAGly1 derivatized with AGH was bound noncovalently to the P site of E. coli 70 S ribosomes, 5-6% on average was photochemically cross-linked to the ribosomal particles in a reaction requiring poly(G,U), irradiation and the presence of the AGH label in the tRNA. Approximately two-thirds of the covalently attached tRNA was associated with 16 S RNA in the 30 S subunit. This material was judged to be in the P site by the criterion of puromycin reactivity. As partial RNAase digestion of the tRNA-16 S RNA complex produced labeled fragments from both 5' and 3' segments of the rRNA, there appeared to be more than one site of cross-linking in the 30 S subunit. The small amount of N-acetylglycyl-tRNAGly1 associated with the 50 S subunit was also linked mainly to rRNA, but it was not puromycin-reactive.