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UDP-N-acetylglucosamine can be bound by pure ribosomes. The part of N-acetylglucosamine-1-P can be transferred from the complex ribosome-UDP-N-acetylglucosamine onto dolichol phosphate. Evidence is presented that N-acetylglucosamine bound to dolichol phosphate can be transferred to the nascent peptide synthesized on the ribosome.
The amino acid sequence of the rat 40 S ribosomal subunit protein S5 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed by the determination, directly from the protein, of 17 residues near the NH2 terminus. S5 has 204 amino acids; the molecular weight is 22,863. The protein designated S5a has the same amino acid sequence as S5 except that it lacks the NH2-terminal 5 residues. It is not known whether the conversion of a portion of S5 to S5a is physiological or fortuitous. The mRNA for S5 has about 820 nucleotides. Hybridization of the S5 cDNA to digests of nuclear DNA indicates that the rat genome has only a single copy of the gene; this is in distinction to the mouse and human genomes which have three to six copies of the S5 gene. Rat ribosomal protein S5 is related to the eubacteria, the arachaebacteria, and the chloroplast family of S7 ribosomal proteins. There is a peptide of 16 residues at the carboxyl terminus of S5 that is highly conserved in 18 species spanning the three kingdoms and chloroplasts.
The sequences of two ribosomal proteins from two widely divergent species of archaebacteria, Halobacterium cutirubrum and Sulfolobus solfataricus, have been deduced from the structure of their respective genes. These two proteins were found to be equivalent to the L11 and L1 ribosomal proteins of the eubacterium Escherichia coli. Sequence comparison revealed that the archaebacterial L11e (equivalent to E. coli L11) proteins are longer than the eubacterial protein due to a C-terminal extension of about 30 residues. The archaebacterial L11e proteins, like the E. coli L11, are rich in proline residues; most of these are conserved. L11 is the most highly methylated protein in the E. coli ribosome. However, sites of methylation are generally not conserved in the archaebacterial L11e proteins. The region of highest sequence similarity between L11 and the archaebacterial L11e proteins is the N-terminal domain. This domain is believed to interact with release factor 1 during termination of translation. The amino acid sequences of the archaebacterial L1e proteins were compared to the eubacterial E. coli L1 and Bacillus stearothermophilus L1e sequences. The archaebacterial L1e proteins are slightly shorter at both their N- and C-termini. A region of high sequence similarity (7 of 14 residues) occurs near the center of the proteins.
Addition of initiation factor IF3 to solutions of E. coli ribosomes dramatically alters their behavior in pressure-jump relaxation kinetic experiments in which 90 degrees light-scattering is used to monitor the macromolecular reaction. The effect of IF3 on relaxation processes attributed to "tight" couples is strongly dependent on the Mg2+ concentration. At 2.5 mM Mg2+, addition of 1 molar equivalent of IF3 decreases the relaxation amplitude by a factor of 3 relative to ribosome solutions without IF3. However, at 5.0 mM Mg2+, addition of 1 molar equivalent of IF3 produces a marked increase in the relaxation amplitude, by a factor of 2-8 fold relative to ribosomes in the absence of IF3. IF3 has no effect on the relaxation process attributed to "loose" couples at 10 mM Mg2+. While we are unable to propose a precise mechanism for IF3 action with the data on hand, our results require that the 30S . IF3 complex either reacts with the 50S subunit, forming a 70S . IF3 intermediate, or acts as a pool of reactive 30S subunit. Further kinetic evidence is required to distinguish between these possible pathways.
It is shown that preparations of EF-T are practically free from GTPase activity, if they were formed from EF-Tu and EF-Ts carefully purified from GTPase beforehand. Some Ef-T-dependent GTPase can arise after addition of tRNA or aminoacyl-tRNA preparations to a EF-T+ribosomes mixture. Poly(U) stimulates, as a rule, EF-T-dependent GTPase in the EF-T/ribosomes+tRNA mixture. Uncoupled EF-T-dependent GTPase of the EF-T+ribosomes+tRNA+poly(U) mixture is by 1--2 orders of magnitude less than uncoupled EF-G-dependent GTPase under the same conditions. A conclusion is made that uncoupled EF-T-dependent GTPase is an inevitable obstacle when studying the GTP expenditure in the process of ribosomal protein synthesis, if a traditional cell-free protein-synthesizing system is used for this purpose.
A variety of compounds related to the 5'-terminal "cap" (m7GpppN) of eukaryotic mRNA's were chemically synthesized and tested as inhibitors of reovirus mRNA binding to wheat germ ribosomes. Under our conditions of mRNA binding to ribosomes, 7-methyl-, 7-ethyl-, and 7-benzyl-GDP, but not GDP, decreased stable initiation complex formation by 70 to 80% at a concentration of 0.1 mM indicating that 7-substitution, but not a specific substituent, was required for the effect. Elimination of the positive charge on the imidazole of the 7-substituted compounds by treatment with alkali destroyed their inhibitory activity. Similarly, reduction to 8-hydro-m7GDP reversibly decreased the activity of m7GDP. The results were consistent with the hypothesis that the positive charge resulting from 7-alkylation provides an active cap conformer for binding via interaction of phosphate oxygens with the positively charged imidazole moiety. In accord with this suggestion, 7-carboxymethyl GDP and 7,8-dimethyl GDP were found to be less inhibitory than m7GDP. A 2-amino group was also important since m7IDP was less effective than m7GDP and 0.1 mM m7XDP did not inhibit ribosome binding. Other poor inhibitors were 6-Cl-m7GDP and 1,7-dimethyl GDP but N2,7-dimethyl GDP, 2'-deoxy-m7GDP, and m7GppI had essentially the same activity as m7GDP.
We have generated a computerized fit between the 3-dimensional map of the E.coli 30S ribosomal proteins, as determined by neutron scattering, and the recently published 3-dimensional model for the 16S RNA. To achieve this, the framework of coordinates for RNA-protein cross-link sites on the phosphate backbone in the RNA model was related to the corresponding framework of coordinates for the mass centres of the proteins by a least squares fitting procedure. The resulting structure, displayed on a computer graphics system, gives the first complete picture of the E.coli 30S ribosomal subunit showing both the proteins and the double-helical regions of the RNA. The root mean square distance between cross-link sites and protein centres is 32 A. The position of the mass centre of the combined double-helical regions was calculated from the model and compared with the position of the mass centre of the complete set of proteins. The two centres are displaced relative to one another by 20 A in the model structure, in good agreement with the experimental value of 25 A found by neutron scattering.
We have constructed complexes of ribosomal proteins S8, S15, S8 + S15 and S8 + S15 + S6 + S18 with 16 S ribosomal RNA, and probed the RNA moiety with a set of structure-specific chemical and enzymatic probes. Our results show the following effects of assembly of proteins on the reactivity of specific nucleotides in 16 S rRNA. (1) In agreement with earlier work, S8 protects nucleotides in and around the 588-606/632-651 stem from attack by chemical probes; this is supported by protection in and around these same regions from nucleases. In addition, we observe protection of positions 573-575, 583, 812, 858-861 and 865. Several S8-dependent enhancements of reactivity are found, indicating that assembly of this protein is accompanied by conformational changes in 16 S rRNA. These results imply that protein S8 influences a much larger region of the central domain than was previously suspected. (2) Protein S15 protects nucleotides in the 655-672/734-751 stem, in agreement with previous findings. We also find S15-dependent protection of nucleotides in the 724-730 region. Assembly of S15 causes several enhancements of reactivity, the most striking of which are found at G664, A665, G674, and A718. (3) The effects of proteins S6 and S18 are dependent on the simultaneous presence of both proteins, and on the presence of protein S15. S6 + S18-dependent protections are located in the 673-730 and 777-803 regions. We observed some variability in our results with these proteins, depending on the ratio of protein to RNA used, and in different trials using enzymatic probes, possibly due to the limited solubility of protein S18. Consistently reproducible was protection of nucleotides in the 664-676 and 715-729 regions. Among the latter are three of the nucleotides (G664, G674 and A718) that are strongly enhanced by assembly of protein S15. This result suggests that an S15-induced conformational change involving these nucleotides may play a role in the co-operative assembly of proteins S6 and S18.
We have studied the effect of assembly of ribosomal proteins S7, S9 and S19 on the accessibility and conformation of nucleotides in 16 S ribosomal RNA. Complexes formed between 16 S rRNA and S7, S7 + S9, S7 + S19 or S7 + S9 + S19 were subjected to a combination of chemical and enzymatic probes, whose sites of attack in 16 S rRNA were identified by primer extension. The results of this study show that: (1) Protein S7 affects the reactivity of an extensive region in the lower half of the 3' major domain. Inclusion of proteins S9 or S19 with S7 has generally little additional effect on S7-specific protection of the RNA. Clusters of nucleotides that are protected by protein S7 are localized in the 935-945 region, the 950/1230 stem, the 1250/1285 internal loop, and the 1350/1370 stem. (2) Addition of protein S9 in the presence of S7 causes several additional effects principally in two structurally distal regions. We observe strong S9-dependent protection of positions 1278 to 1283, and of several positions in the 1125/1145 internal loop. These findings suggest that interaction of protein S9 with 16 S rRNA results in a structure in which the 1125/1145 and 1280 regions are proximal to each other. (3) Most of the strong S19-dependent effects are clustered in the 950-1050 and 1210-1230 regions, which are joined by base-pairing in the 16 S rRNA secondary structure. The highly conserved 960-975 stemp-loop, which has been implicated in tRNA binding, appears to be destabilized in the presence of S19. (4) Protein S7 causes enhanced reactivity at several sites that become protected upon addition of S9 or S19. This suggests that S7-induced conformational changes in 16 S rRNA play a role in the co-operativity of assembly of the 3' major domain.
We have isolated several new clones of human ribosomal DNA. Each clone contains part of the external transcribed spacer, a complete 18 S-rRNA gene, the internal transcribed spacers, a complete 28 S-rRNA gene and a short downstream flanking region. We present a detailed map of the human ribosomal transcription unit with the locations of numerous useful restriction sites. In particular, a unique NheI site in the 5.8 S-rRNA gene enabled this gene to be mapped with respect to the 18 S-rRNA and 28 S-rRNA genes. The human 45 S-rRNA coding region is approx. 13,000 nucleotide residues long, of which the external transcribed spacer comprises approx. 3700 nucleotide residues and the first and second internal transcribed spacers comprise approx. 1070 and 1200 nucleotide residues respectively. A partial survey for sites of variation between clones has revealed a single point of variation among 18 S-rRNA gene sequences (a T/C variation at position 140), several sites of length variation in the regions of the transcribed spacers closely flanking the 18 S-rRNA genes, and some sites of length variation among 28 S-rRNA genes. Most of these sites of variation are associated with simple sequence tracts and are in regions that are known to undergo relatively rapid evolutionary divergence. In particular, the sites of variation among 28 S-rRNA genes occur in G + C-rich tracts whose lengths vary among vertebrates and that can be correlated with extensive hairpin structures previously observed by electron microscopy. Each of the clones so far surveyed in detail differs from the others in one or more respects.
A reconstruction, at 40 A, of the Escherichia coli ribosome imaged by cryo-electron microscopy, obtained from 303 projections by a single-particle method of reconstruction, shows the two subunits with unprecedented clarity. In the interior of the subunits, a complex distribution of higher mass density is recognized, which is attributed to ribosomal RNA. The masses corresponding to the 16S and 23S components are linked in the region of the platform of the small subunit. Thus the topography of the rRNA regions responsible for protein synthesis can be described.
A three-dimensional reconstruction of the eukaryotic 80S monosome from a frozen-hydrated electron microscopic preparation reveals the native structure of this macromolecular complex. The new structure, at 38A resolution, shows a marked resemblance to the structure determined for the E. coli 70S ribosome (Frank, J., A. Verschoor, Y. Li, J. Zhu, R.K. Lata, M. Radermacher, P. Penczek, R. Grassucci, R.K. Agrawal, and Srivastava. 1996b. In press; Frank, J., J. Zhu, P. Penczek, Y. Li, S. Srivastava ., A. Verschoor, M. Radermacher, R. Grassucci, R.K. Lata, and R. Agrawal. 1995. Nature (Lond.).376:441-444.) limited to a comparable resolution, but with a number of eukaryotic elaborations superimposed. Although considerably greater size and intricacy of the features is seen in the morphology of the large subunit (60S vs 50S), the most striking differences are in the small subunit morphology (40S vs 30S): the extended beak and crest features of the head, the back lobes, and the feet. However, the structure underlying these extra features appears to be remarkably similar in form to the 30S portion of the 70S structure. The intersubunit space also appears to be strongly conserved, as might be expected from the degree of functional conservation of the ribosome among kingdoms (Eukarya, Eubacteria, and Archaea). The internal organization of the 80S structure appears as an armature or core of high-density material for each subunit, with the two cores linked by a single bridge between the platform region of the 40S subunit and the region below the presumed peptidyltransferase center of the 60S subunit. This may be equated with a close contact of the 18S and 28S rRNAs in the translational domain centered on the upper subunit:subunit interface.
The ribosomal protein complex L8 of Escherichia coli consists of two dimers of protein L7/L12 and one monomer of protein L10. This pentameric complex and ribosomal protein L11 bind in mutually cooperative fashion to 23 S rRNA and protect specific fragments of the latter from digestion with ribonuclease T1. Oligonucleotides protected either by the L8 complex alone or by the complex plus protein L11 were isolated from such digests and shown to rebind specifically to these proteins. They were also subjected to nucleotide sequence analysis. The longest oligonucleotide, protected by the L8 complex alone, consisted of residues 1028-1124 of 23 S rRNA and included all the other RNA fragments produced in this study. Previously, protein L11 had been shown to protect residues 1052-1112 of 23 S rRNA. It is concluded that the binding sites for the L8 protein complex and for protein L11 are immediately adjacent within 23 S rRNA of E. coli.
L15, a 15 kDa protein of the large ribosomal subunit, interacts with over ten other proteins during 50 S assembly in vitro. We have probed the interaction L15 with 23 S rRNA in 50 S ribosomal subunits by chemical footprinting, and have used localized hydroxyl radical probing, generated from Fe(II) tethered to unique sites of L15, to characterize the three-dimensional 23 S rRNA environment of L15. Footprinting of L15 was done by reconstituting purified, recombinant L15 with core particles derived from Escherichia coli 50 S subunits by treatment with 2 M LiCl. The cores migrate as compact 50 S-like particles in sucrose gradients, contain 23 S and 5 S rRNA, and lack a subset of the 50 S proteins, including L15. Using both Fe(II).EDTA and dimethyl sulfate, we have identified a strong footprint for L15 in the region spanning nucleotides 572-654 in domain II of 23 S rRNA. This footprint cannot be detected when L15 is incubated with "naked" 23 S rRNA, indicating that formation of the L15 binding site requires a partially assembled particle.Protein-tethered hydroxyl radical probing was done using mutants of L15 containing single cysteine residues at amino acid positions 68, 71 and 115. The mutant proteins were derivatized with 1-[p-(bromo-acetamido)benzyl]-EDTA. Fe(II), bound to core particles, and hydroxyl radical cleavage was initiated. Distinct but overlapping sets of cleavages were obtained in the footprinted region of domain II, and in specific regions of domains I, IV and V of 23 S rRNA. These data locate L15 in proximity to several 23 S rRNA elements that are dispersed in the secondary structure, consistent with its central role in the latter stages of 50 S subunit assembly. Furthermore, these results indicate the proximity of these rRNA regions to one another, providing constraints on the tertiary folding of 23 S rRNA.
Domain III of Saccharomyces cerevisiae 25 S rRNA contains the recognition site for the primary rRNA-binding ribosomal protein L25, which belongs to the functionally conserved EL23/L25 family of ribosomal proteins. The EL23/L25 binding region is very complex, consisting of several irregular helices held together by long-distance secondary and tertiary interactions. Moreover, it contains the eukaryote-specific V9 (D7a) expansion segment. Functional characterisation of the structural elements of this site by a detailed in vitro and in vivo mutational analysis indicates the presence of two separate regions that are directly involved in L25 binding. In particular, mutation of either of two conserved nucleotides in the loop of helix 49 significantly reduces in vitro L25 binding, thus strongly supporting their role as attachment sites for the r-protein. Two other helices appear to be primarily required for the correct folding of the binding site. Mutations that abolish in vitro binding of L25 block accumulation of 25 S rRNA in vivo because they stall pre-rRNA processing at the level of its immediate precursor, the 27 S(B) pre-rRNA. Surprisingly, several mutations that do not significantly affect L25 binding in vitro cause the same lethal defect in 27 S(B) pre-rRNA processing. Deletion of the V9 expansion segment also leads to under-accumulation of mature 25 S rRNA and a twofold reduction in growth rate. We conclude that an intact domain III, including the V9 expansion segment, is essential for normal processing and assembly of 25 S rRNA.
Selected strains of Saccharomyces cerevisiae were mutagenized with nitrosoguanidine and temperature-sensitive mutants isolated. These mutants were screened by two-dimensional gel-electrophoresis for the presence of ribosomal proteins with altered mobility relative to parental preparations. Electrophoretic changes were detected in three mutants designated ts205, ts212 and ts417, with the alterations apparently the same in the three cases. All three mutants were more sensitive than were their parents to the antibiotics G418, hygromycin B and MDMP. Mutant ts212 has an abnormal distribution of native ribosomal subunits and appears to be defective in its assembly of the smaller subparticle.
The E. coli 16 S rRNA with single-site breaks centered at position 777 or 785 was obtained by RNase H site-specific cleavage of rRNA. Spontaneous dissociation of the cleaved 16 S rRNA into fragments occurred under 'native' conditions. The reassociation of the 16 S rRNA fragments was possible only in the presence of ribosomal proteins. The combination of S4 and S16(S17) ribosomal proteins interacting mainly with the 5'-end domain of 16 S rRNA was sufficient for reassociation of the fragments. The 30 S subunits with fragmented RNA at ca. 777 region retained some poly(U)-directed protein synthetic activity.
The type 1 ribosome-inactivating protein (RIP) saporin 5 isolated from seeds of Saponaria officinalis L. strongly inhibited translation carried out by Vicia sativa L. purified ribosomes. The toxin multidepurinated V. sativa rRNA, which upon treatment with acid aniline releases several RNA fragments including an RNA fragment of approximately 370 nucleotides the 5'-end sequence of which was 5'-GAGGAACG-3'.