Evidence for a tRNA/rRNA interaction site within the peptidyltransferase center of the Escherichia coli ribosome

A nine-base oligodeoxyribonucleotide complementary to bases 2497-2505 of 23S rRNA was hybridized to both 50S subunits and 70S ribosomes. The binding of the probe to the ribosome or ribosomal subunits was assayed by nitrocellulose filtration and by sucrose gradient centrifugation techniques. The location of the hybridization site was determined by digestion of the rRNA/cDNA heteroduplex with ribonuclease H and gel electrophoresis of the digestion products, followed by the isolation and sequencing of the smaller digestion fragment. The cDNA probe was found to interact specifically with its rRNA target site. The effects on probe hybridization to both 50S and 70S ribosomes as a result of binding deacylated tRNA(Phe) were investigated. The binding of deacylated tRNA(Phe), either with or without the addition of poly(uridylic acid), caused attenuation of probe binding to both 50S and 70S ribosomes. Probe hybridization to 23S rRNA was decreased by about 75% in both 50S subunits and 70S ribosomes. These results suggest that bases within the 2497-2505 site may participate in a deacylated tRNA/rRNA interaction.     

Identification of 50S components neighboring 23S rRNA nucleotides A2448 and U2604 within the peptidyl transferase center of Escherichia coli ribosomes

The 23S rRNA nucleotides 2604-12 and 2448-58 fall within the central loop of domain V, which forms a major part of the peptidyl transferase center of the ribosome. We report the synthesis of radioactive, photolabile 2'-O-methyloligoRNAs, PHONTs 1 and 2, complementary to these nucleotides and their exploitation in identifying 50S ribosomal subunit components neighboring their target sites. Photolysis of the 50S complex with PHONT 1, complementary to nts 2604-12, leads to target site-specific photoincorporation into protein L2 and 23S rRNA nucleotides A886, Alpha1918, A1919, G1922-C1924, U2563, U2586, and C2601. Photolysis of the 50S complex with PHONT 2, complementary to nts 2448-58, leads to target site-specific probe photoincorporation into proteins L2, L3, one or more of proteins L17, L18, L21, and of proteins L9, L15, L16, and 23S rRNA nucleotides C2456 and psi2457. Chemical footprinting studies show that 2'-O-methyloligoRNA binding causes little distortion of the peptidyl transferase center but do provide suggestive evidence for the location of flexible regions within 23S rRNA. The significance of these results for the structure of the peptidyl transferase center is considered.     

Identification of a rRNA/chloramphenicol interaction site within the peptidyltransferase center of the 50 S subunit of the Escherichia coli ribosome.

We have used oligodeoxyribonucleotide probes to investigate possible interactions between chloramphenicol and portions of the rRNA contained within the peptidyltransferase center of the Escherichia coli ribosome. Oligodeoxyribonucleotide probes complementary to bases 2448-2454, 2468-2482, and 2497-2505 of 23 S rRNA were hybridized to 50 S subunits in situ. Probe binding was qualitatively assessed by sucrose gradient centrifugation. Each probe was shown to bind specifically with its intended binding site through digestion of the rRNA within the RNA/DNA hetero-duplexes with RNase H and analysis of the digestion fragments using gel electrophoresis. Competitive binding experiments were conducted between each probe and the antibiotics chloramphenicol and erythromycin. The binding of a probe complementary to bases 2497-2505 was attenuated by 70% upon the binding of chloramphenicol. A probe complementary to bases 2468-2482 showed an increase in binding of 14% while binding of a probe complementary to bases 2448-2454 was not affected by chloramphenicol binding. Erythromycin did not affect the binding of any of these probes to 50 S subunits. These results suggest that bases within the 2497-2505 region of 23 S rRNA in E. coli may be involved in a chloramphenicol/rRNA interaction.     

Placement of the alpha-sarcin loop within the 50S subunit: evidence derived using a photolabile oligodeoxynucleotide probe.

We report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe and its exploitation in identifying 50S ribosomal subunit components neighboring the alpha-sarcin loop. The probe is complementary to 23S rRNA nt 2653-2674. Photolysis of the complex formed between the probe and 50S subunits leads to site-specific probe photoincorporation into proteins L2, the most highly labeled protein, L1, L15, L16 and L27, labeled to intermediate extents, and L5, L9, L17 and L24, each labeled to a minor extent. Portions of each of these proteins thus lie within 23 A of nt U2653. These results lead us to conclude that the alpha-sarcin loop is located at the base of the L1 projection within the 50S subunit. Such placement, near the peptidyl transferase center, provides a rationale for the extreme sensitivity of ribosomal function to cleavage of the alpha-sarcin loop.     

In vitro incorporation of eubacterial, archaebacterial and eukaryotic 5S rRNAs into large ribosomal subunits of Bacillus stearothermophilus.

Bacillus stearothermophilus large ribosomal subunits were reconstituted in the presence of 5S rRNAs from different origins and tested for their biological activities. The results obtained have shown that eubacterial and archaebacterial 5S rRNAs can easily substitute for B. stearothermophilus 5S rRNA in the reconstitution, while eukaryotic 5S rRNAs yield ribosomal subunits with reduced biological activities. From our results we propose an interaction between nucleotides 42-47 of 5S rRNA and nucleotides 2603-2608 of 23S rRNA during the assembly of the 50S ribosomal subunit. Other experiments with eukaryotic 5.8S rRNAs reveal, if at all, a very low incorporation of these RNA species into the reconstituted ribosomes.     

Identification of 23S rRNA nucleotides neighboring the P-loop in the Escherichia coli 50S subunit.

We report the synthesis of a radioactive, photolabile 2'-O-methyloligoRNA probe, 2258-53/52(SAz)-48, PHONT1, and its exploitation in identifying 23S rRNA nucleotides neighboring the so-called 'P-loop'. The probe is complementary to nt 2248-2258 in Escherichia coli 50S subunits. PHONT1 contains a p-azidophenacyl group attached to a phosphorothioate bridge between the nucleotides complementary to the positions 2252-2253, such that the photogenerated nitrene is maximally 17-19 A from 23S RNA nucleotides G2252 and G2253. PHONT1 binds to the 50S subunit, and photoincorporates within or immediately adjacent to its target site, as well as into several nucleotides falling between G2357 and A2430. The significance of these results for the structure of the peptidyl transferase center is considered. The PHONT approach is generally applicable to studies of complex RNA-containing molecules.     

Blocking of the initiation of protein biosynthesis by a pentanucleotide complementary to the 3' end of Escherichia coli 16 S rRNA.

Hydrogen bonding between the 3' terminus of 16 S rRNA (... C-A-C-C-U-C-C-U-U-A-OH3) and complementary sequences within the initiator region of mRNA may be a crucial event in the specific initiation of protein biosynthesis (Shine, J., and Dalgarno, L. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 1342-1346; Steitz, J. A., and Jakes, K. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4734-4738). Using equilibrium dialysis, we have studied the binding of G-A-dG-dG-U (which is complementary to the 3' end of 16 S rRNA and which has been synthesized enzymatically) to initiation factor-free Escherichia coli ribosomes. We have also investigated the effects of the pentanucleotide on initiation reactions in E. coli ribosomes. G-A-dG-dG-U has a specific binding site on the 30 S ribosome with an association constant of 2 x 10(6) M-1 at 0 degrees C. G-A-dG-dG-U inhibits the R17 mRNA-dependent binding of fMet-tRNA by about 70%, both with 70 S ribosomes and 30 S subunits. In contrast, the A-U-G-dependent initiation reaction and the poly(U)-dependent Phe-tRNA binding was not affected by the pentanucleotide with both ribosomal species.     

Identification by RNA-protein cross-linking of ribosomal proteins located at the interface between the small and the large subunits of mammalian ribosomes

Protein constituents at the subunit interface of rat liver ribosomes were analysed by cross-linking with the bifunctional reagent, diepoxybutane (distance between reactive groups 4 A). Isolated 40S and 60S subunits were labelled with 125I and recombined with unlabelled complementary subunits. The two kinds of selectively labelled 80S ribosomes were treated with diepoxybutane at low concentration. Radioactive ribosomal proteins covalently attached to the rRNA of the unlabelled complementary subparticles were isolated by repeated gradient centrifugation. The RNA-bound, labelled proteins were identified by two-dimensional gel electrophoresis. The experiments showed that proteins S2, S3, S4, S6, S7, S13, and S14 in the small subunit of rat liver ribosomes are located at the ribosomal interface in close proximity to 28S rRNA. Similarly, proteins L3, L6, L7, and L8 were found at the the interface of the large ribosomal subunit in the close vicinity of 18S rRNA.     

Reconstitution of Escherichia coli ribosomes containing puromycin-modified S14: functional effects of the photoaffinity labeling of a protein essential for tRNA binding

In previous work we have shown that puromycin photoaffinity labels two proteins, L23 and S14, from separate sites of high affinity on Escherichia coli ribosomes [Jaynes, E. N., Jr., Grant, P. G., Giangrande, G., Wieder, R., & Cooperman, B. S. (1978) Biochemistry 17, 561-569; Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274], that puromycin-modified S14 is separable from native S14 by reverse-phase high-performance liquid chromatography (RP-HPLC), and that ribosomal proteins prepared by RP-HPLC can be reconstituted into active 30S subunits [Kerlavage, A. R., Weitzmann, C. J., & Cooperman, B. S. (1984) J. Chromatogr. 317, 201-212]. In this work we definitively identify puromycin-modified S14 by tryptic fingerprinting, an analysis that also provides evidence that the single tryptophan-containing peptide in S14 is the site of puromycin photoincorporation. We show that reconstituted 30S subunits, in which all of the S14 present is stoichiometrically modified with puromycin and all other ribosomal components are present in unmodified form, lack Phe-tRNAPhe binding activity and further that 70S ribosomes containing such reconstituted 30S subunits have substantially diminished binding activity to both the A and P sites, as differentiated through use of tetracycline. Suitable control experiments strongly indicate that this loss of activity is a direct consequence of puromycin photoincorporation.     

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.     

Localization of spermine binding sites in 23S rRNA by photoaffinity labeling: parsing the spermine contribution to ribosomal 50S subunit functions

Polyamine binding to 23S rRNA was investigated, using a photoaffinity labeling approach. This was based on the covalent binding of a photoreactive analog of spermine, N¹-azidobenzamidino (ABA)-spermine, to Escherichia coli ribosomes or naked 23S rRNA under mild irradiation conditions. The cross-linking sites of ABA-spermine in 23S rRNA were determined by RNase H digestion and primer-extension analysis. Domains I, II, IV and V in naked 23S rRNA were identified as discrete regions of preferred cross-linking. When 50S ribosomal subunits were targeted, the interaction of the photoprobe with the above 23S rRNA domains was elevated, except for helix H38 in domain II whose susceptibility to cross-linking was greatly reduced. In addition, cross-linking sites were identified in domains III and VI. Association of 30S with 50S subunits, poly(U), tRNA^Phe and AcPhe-tRNA to form a post-translocation complex further altered the cross-linking, in particular to helices H11–H13, H21, H63, H80, H84, H90 and H97. Poly(U)-programmed 70S ribosomes, reconstituted from photolabeled 50S subunits and untreated 30S subunits, bound AcPhe-tRNA in a similar fashion to native ribosomes. However, they exhibited higher reactivity toward puromycin and enhanced tRNA-translocation efficiency. These results suggest an essential role for polyamines in the structural and functional integrity of the large ribosomal subunit.     

Putative implication of 3′-terminal segment of 18S rRNA in translation initiation of uncapped mRNAs in plants

A putative implication 3′-terminal 18S rRNA segment in the cap-independent initiation of translation on plant ribosomes was studied. It was shown that 3′-terminal segment (nucleotides 1777–1811) of 18S rRNA including the last hairpin 45 was accessible for complementary interactions within 40S ribosomal subunits. Oligonucleotides complementary to this segment of rRNA, when added to wheat germ cell-free protein synthesizing system, specifically inhibited translation of uncapped reporter mRNA encoding β-glucuronidase. In the 5′-untranslated region (UTR), the reporter mRNA contained a leader sequence of potato virus Y (PVY) genomic RNA with fragments complementary to the region 1777–1811. A sequence corresponding to nucleotides 291–316 of PVY, which was complementary to most of the 3′-terminal 18S rRNA segment 1777–1808, was shown to enhance translational efficiency of the reporter mRNAs when placed into 5′-UTR. The obtained results suggest that complementary interactions between 5′-UTR of mRNA and 3′-terminal segment of 18S rRNA can take place during cap-independent translation initiation.     

Changes in the conformation of 5S rRNA cause alterations in principal functions of the ribosomal nanomachine

5S rRNA is an integral component of the large ribosomal subunit in virtually all living organisms. Polyamine binding to 5S rRNA was investigated by cross-linking of N¹-azidobenzamidino (ABA)-spermine to naked 5S rRNA or 50S ribosomal subunits and whole ribosomes from Escherichia coli cells. ABA-spermine cross-linking sites were kinetically measured and their positions in 5S rRNA were localized by primer extension analysis. Helices III and V, and loops A, C, D and E in naked 5S rRNA were found to be preferred polyamine binding sites. When 50S ribosomal subunits or poly(U)-programmed 70S ribosomes bearing tRNA^Phe at the E-site and AcPhe-tRNA at the P-site were targeted, the susceptibility of 5S rRNA to ABA-spermine was greatly reduced. Regardless of 5S rRNA assembly status, binding of spermine induced significant changes in the 5S rRNA conformation; loop A adopted an apparent ‘loosening’ of its structure, while loops C, D, E and helices III and V achieved a more compact folding. Poly(U)-programmed 70S ribosomes possessing 5S rRNA cross-linked with spermine were more efficient than control ribosomes in tRNA binding, peptidyl transferase activity and translocation. Our results support the notion that 5S rRNA serves as a signal transducer between regions of 23S rRNA responsible for principal ribosomal functions.     

Large-scale motions within ribosomal 50S subunits as demonstrated using photolabile oligonucleotides

Photolabile oligonucleotides (PHONTs) bind to rRNA sequences to which they are complementary and, on photolysis, incorporate into neighboring ribosomal components. Here we report on photocrosslinking results obtained with PHONTs targeting 23S rRNA nucleotides 1882-1892, in the long lateral arm of the 50S subunit (PHONT 1892), and 1085-1093, in the L11 binding domain (PHONT 1093). Photolysis of the PHONT 1892.50S and PHONT 1093.50S complexes leads to formation of 'long-range' crosslinks from C1892 to U1094/A1095 and G1950, and from G1093 to U1712/1716 and U1926, that are clearly incompatible with published crystal structures of 50S subunits. These results provide strong evidence that within the 50S subunit (a) the L11 binding domain can extend in an arm-like fashion, accessing large areas of the ribosome, and (b) the lateral arm can bend about the noncanonical helix at its center. Such motions may have functional relevance in identifying regions that undergo major conformational change as the ribosome moves through its catalytic cycle.     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

Interaction of tetracycline with RNA: photoincorporation into ribosomal RNA of Escherichia coli.

Photolysis of [3H]tetracycline in the presence of Escherichia coli ribosomes results in an approximately 1:1 ratio of labelling ribosomal proteins and RNAs. In this work we characterize crosslinks to both 16S and 23S RNAs. Previously, the main target of photoincorporation of [3H]tetracycline into ribosomal proteins was shown to be S7, which is also part of the one strong binding site of tetracycline on the 30S subunit. The crosslinks on 23S RNA map exclusively to the central loop of domain V (G2505, G2576 and G2608) which is part of the peptidyl transferase region. However, experiments performed with chimeric ribosomal subunits demonstrate that peptidyltransferase activity is not affected by tetracycline crosslinked solely to the 50S subunits. Three different positions are labelled on the 16S RNA, G693, G1300 and G1338. The positions of these crosslinked nucleotides correlate well with footprints on the 16S RNA produced either by tRNA or the protein S7. This suggests that the nucleotides are labelled by tetracycline bound to the strong binding site on the 30S subunit. In addition, our results demonstrate that the well known inhibition of tRNA binding to the A-site is solely due to tetracycline crosslinked to 30S subunits and furthermore suggest that interactions of the antibiotic with 16S RNA might be involved in its mode of action.     

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.     

Structural analysis of the peptidyl transferase region in ribosomal RNA of the eukaryote Xenopus laevis

Accessible single-strand bases in Xenopus laevis 28 S ribosomal RNA (rRNA) Domain V, the peptidyl transferase region, were determined by chemical modification with dimethylsulfate, 1-cyclohexyl-3-(2-morpholinoethyl-carbodiimide metho-p-toluene sulfonate and kethoxal, followed by primer extension. The relative accessibilities of three rRNA substrates were compared: deproteinized 28 S rRNA under non-denaturing conditions (free 28 S rRNA), 60 S subunits and 80 S ribosomes. Overall, our experimental results support the theoretical secondary structure model of Domain V derived by comparative sequence analysis and compensatory base-pair changes, and support some theoretical tertiary interactions previously suggested by covariation. The 60 S subunits and 80 S ribosomes generally show increasing resistance to chemical modification. Bases which are sensitive in free 28 S rRNA but protected in 60 S subunits may be sites for ribosomal protein binding or induced structural rearrangements. Another class of nucleotides is distinguished by its sensitivity in 60 S subunits but protection in 80 S ribosomes; these nucleotides may be involved in subunit-subunit interactions or located at the interface of the ribosome. We found a third class of bases, which is protected in free 28 S rRNA but sensitive in 60 S subunits and/or 80 S ribosomes, suggesting that structural changes occur in Domain V as a result of subunit assembly and ribosome formation. One such region is uniquely hypersensitive in eukaryotic ribosomes but is absent in Escherichia coli ribosomes. Sites that we determined to be accessible on empty 80 S ribosomes could serve as recognition sites for translation components.