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A new ribosomal mutant resistant to erythromycin is described. The product of the gene eryC seems to be implicated in the assembly of the two ribosomal subunits, particularly in the maturation process of the RNA 23S and 16S.
The exchange of ribosomal proteins among ribosomes of E. coli has been measured, using a density label technique. As expected most of the proteins do not exchange appreciably. However a substantial fraction of each of proteins S1, S2, S21, L7/L12, L9, L10, L11, L26 and L33 is found to exchange, but exchange of S1, S2, L7/L12, L10, L11 and L26 is found to occur in vitro after lysis of the cells, and therefore it is not possible to say whether or not these proteins also exchange in vivo. tin contrast S21, l9 and L33 do not exchange after lysis of the cells and we therefore conclude that these proteins exchange in vivo. The maximum level of exchange of S21, L9 and L33 is attained so rapidly that we were unable to show whether or not it was dependent on protein synthesis.
The cytoplasmic 80s ribosomal proteins from the cells of yeast Sachharomyces cerevisiae were analysed by SDS two-dimensional polyacrylamide gel electrophoresis. Seventyfour proteins were identified and consecutively numbered from 1 to 74. Upon oxidation of the 80s proteins with performic acid, ten proteins (no. 15, 20, 35, 40, 44, 46, 49, 51, 54 and 55) were dislocated on the gel without change of the total number of protein spots. Five proteins (no. 8, 14, 16, 36 and 74) were phosphorylated in vivo as seen in 32P-labelling experiments. The large and small subunits separated in low magnesium medium were analyzed by the above gel electrophoresis. At least forty-five and twenty-eight proteins were assumed to be in the large and small subunits, respectively. All proteins found in the 80s ribosomes, except for no. 3, were detected in either subunit without appearance of new spots. The acidic protein no. 3 seems to be lost during subunit dissociation.
Derivatives of isogenic stringent (relA+) and relaxed (relA) strains of Escherichia coli were compared in respect of rates of the dissociation of peptidyl-tRNA from ribosomes during protein synthesis. The derivatives both contained a mutant pth gene which rendered temperature-sensitive their peptidyl-tRNA hydrolase (E.C. 3.1.1.29) activities. After shifting from permissive 30 degrees C to non-permissive 40 degrees C, dissociated peptidyl-tRNA accumulated and was assayed chemically or by its cytotoxic effects. In unperturbed (except for the temperature shift) cultures the relA strain accumulated peptidyl-tRNA significantly more slowly than did its relA+ isogenic cousin. Both strains responded approximately equally to non-lethal doses of erythromycin or to starvation for amino acids. Both these perturbations enhanced the dissociation and accumulation of peptidyl-tRNA. While growing at 30 degrees C, both strains responded significantly to a nutritional downshift from growth in medium containing glucose plus amino acids to growth in medium containing only amino acids. Taken together the results suggested that different intracellular concentrations of ppGpp in unperturbed cells, attributable to the different relA alleles, could account for the differences in dissociation and accumulation of peptidyl-tRNA. Our observation of a lower rate of dissociation of peptidyl-tRNA in the relA strain, coupled with the reported lower intracellular ppGpp and lower accuracy of protein synthesis, is consistent with the idea that relA strains have less efficient ribosomal editing of erroneous peptidyl-tRNA.
Among the mitochondrial conditional mutations localized in the gene coding for the 21S ribosomal RNA, one--ts 902--produces severely reduced amounts of 21S RNA and 50S subunit. We investigated its physiological properties and found that this thermosensitive mutation was associated with highly pleiotropic effects. The mutant phenotype is associated with cell death in certain conditions, and with a massive accumulation of rho- mutants at non-permissive temperature. Furthermore, interactions with the sites of action of erythromycin and chloramphenicol, both localized within the 21S rRNA, were detected. The mutant is hypersensitive to erythromycin and has a cis-incompatibility with the chloramphenicol-resistant mutation CR321. Ts 902 thus appears to have a dual effect, not only at the ribosomal level but also at a cellular level.
Ribosomal RNA isolated from ribosomes present inside avian myeloblastosis virus (AMV) was characterized by electron microscopy using the formamide-urea spreading technique. The molecular weight and the secondary structures were compared with those of r-RNA and precursor r-NA isolated from host cells, the leukemic myeloblasts. The molecular weight of viral r-RNA (1.62 +/- 0.18 X 10(6) and 0.69 +/- 0.10 X 10(6)) and the molecular weight of cellular r-RNA (1.63 +/- 0.18 X 10(6) and 0.67 +/- 0.09 X 10(6)), the latter obtained from avian myeloblasts, were found to be identical and comparable with the molecular weight of chicken liver r-RNA. Likewise, the secondary structures of viral r-RNA were identical to those of cellular r-RNA. The postulated possible precursor character of viral r-RNA was excluded, since the molecules of viral r-RNA do not show any similarity to those of precursor r-RNA. Previously observed differences in behavior of viral and cellular (myeloblastic) r-RNA in sedimentation and electrophoretic mobility are discussed.
1. Four precursor ribosomal RNAs have been previously characterized in cultured sycamore cells (Acer pseudoplatanus L.). The present paper reports a study of the two smallest ones. 2. (Me-3H)-labelling of the precursor and mature rRNAs has been submitted to kinetic analysis. From pulse-chase experiments it is concluded that the 27S and 20S precursor rRNAs behave like intermediate molecules in the maturation of the 42S and 36S precursor rRNAs into the 26S and 17S components. They are direct precursors to the cytoplasmic and mature rRNAs. 3. Base-composition analyses have been performed on carefully purified molecules. They show that the 27S and 20S precursor rRNAs arise from a single cleavage of the 36S precursor rRNA. In addition, they strongly suggest that the 27S and 20S precursor rRNAs can be taken as specific precursors to the 26S and 17S rRNAs, respectively. These results are reinforced by DNA - RNA hybridization experiments, the measured values being in good agreement with the theoretical ones expected from a specific relationship between the four smallest molecules. Base-compositions of the conserved and non-conserved nucleotide sequences of the primary transcriptional product have been estimated or calculated. 4. On the basis of the present results and those previously reported, a scheme for the biosynthesis of ribosomal RNA in sycamore cells is proposed.
Trypsin covalently bound on collagen membranes has been used to investigate the protein topography in eukaryotic 60S ribosomal subunits. Six proteins are highly exposed to the attack of the immobilized enzyme: L6, L7-L7a, L17, L24, and L31. They are located in two distinct regions, forming two bulges at the ribosomal surface; the first one consists of proteins L6 and L7-L7a, which are screening proteins degraded later, as L4, L14, L23a, and L29; the second one is formed by proteins L17, L24, and L31, which are shielding L19 and L22. L3, L5, L8, L11, L12, L26, L30, L34, and L37a, are located in a trough between the two bulges. L10, close to L5, appears to be more accessible than all these proteins. Several proteins are not degraded by trypsin, even for a very long time of incubation: L9, L13-L13a, L18, L18a, L21, L25, L27-L27a, L28, L32, L35, L35a, L36-L36a, and L38. The cross-linking data suggest that these latter proteins are mainly protected by the proteins located in the L6-L7-L7a region, and by the 28S RNA. A model of protein topography within the 60S rat liver subunits, based on protein accessibility and cross-linking data, is proposed.
The reaction of [3H]p-nitrophenyl acetate (NPA) or [14C]methyl acetimidate (MAI) with amino groups of ribosomal pade between the reactivity of the proteins in situ in the ribosomal subunit with that of isolated protein mixtures. In the small subunit reactivity compared with the protein mixture was only 10-65% in the case of NPA but 45 to more than 100% in the case of MAI. In the large subunit reactivity to MAI was 10-60% that of the isas a denser structure than the small one. In agreement with earlier experiments with iodoacetamide the proteins S2, 5, 7, 8, 10 and 13 of the small subunit and L15, 17, 20, 24, 25, 27, 29, 33, 34, 35 and 38 in the large subunit are quite accessible while proteins S9, 14, 19, 20, 24, 25, 27, 29 and 30 of the small subunit and L1, 7, 8, 10, 11, 19, 28, 31, and 32 of the large one are relatively inaccessible.
We have analysed the effects of an inhibition of DNA replication by hydroxyurea on the synthesis of ribosomal proteins (r-proteins) and ribosomal RNA (rRNA) in Tetrahymena cells resuming growth after long-term starvation. The coordinate regulation of the synthesis of individual r-proteins and their increased rate of synthesis during refeeding are not impaired by inhibition of DNA replication. Moreover, the presence of hydroxyurea does not prevent an increase in the rate of synthesis of rRNA around 70-80 min after refeeding. Previously, this increase was claimed to be gene dose-dependent. Up to 180 min after refeeding, the synthesis of r-proteins appears to be closely coupled with that of rRNA and proceed in stoichiometric balance, irrespective of whether hydroxyurea is present or not. After 180 min of refeeding in the presence of hydroxyurea, this stoichiometric balance breaks down, and the rate of synthesis of r-proteins clearly exceeds that of the rRNA synthesis.
An electrophoretic system capable of separating 25 S, 23 S, 17.5 S and 16 S ribosomal RNA (rRNA) species was used to study the synthesis and fate of rRNA during amino acid starvation and resupplementation of E. coli relaxed strain KL99. This E. coli relAl strain responded to an amino acid starvation by increasing the rate of synthesis of 25 S and 17.5 S precursor rRNA. When the limiting amino acid was resupplemented, a previously observed 40-fold increase in the cellular guanosine 5'-diphosphate, 3'-diphosphate content [Mol. Gen. Genet. (1983) 192, 5-9] appeared to cause a reduction in new rRNA synthesis. Following amino acid resupplementation, the precursor 25 S and 17.5 S rRNA accumulated during the amino acid starvation were conserved and processed to 23 S and 16 S rRNA species, respectively. This suggests that a modified ribosome assembly scheme involving stable precursor rRNA exists in relAl bacteria during periods of amino acid limitation and resupplementation.
The genes for ribosomal proteins L28 and L33 constitute an operon (rpmBG) in E. coli, but in plant chloroplasts L33 is encoded by the chloroplast DNA and L28 seems to be encoded by the nuclear genome. A 15 kDa protein was isolated from the 50 S subunit of tobacco chloroplast ribosomes and its N-terminal amino acid sequence was determined. A cDNA for this protein was cloned and analyzed. The cDNA encodes a 151 amino acid protein consisting of a predicted transit-peptide of 74 amino acids and a mature protein of 77 amino acids. The mature protein is homologous to E. coli L28, hence we named it chloroplast L28 (CL28). This is the first report on the presence of an E. coli L28-like protein in another organism.
Ribosomal proteins from the eggs of sea urchin, Pseudocentrotus depressus, were analyzed by two-dimensional polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. Thirty different proteins including three acidic proteins were detected in the small subunit. The large subunit was found to contain at least 43 different proteins, 42 basic and one acidic. The ribosomal proteins from the small subunit have molecular weights ranging from 11,900 to 51,700 (average molecular weight: 25,600). The molecular weights of the large subunit proteins range from 12,700 to 76,300 (average molecular weight: 28,700).
The capacity of some Escherichia coli (E. coli) ribosomal proteins to bind to tRNA and to hydrolyse their aminoacylated derivatives has been analysed. The following results were obtained: (1) The basic proteins L2, L16 and L33 and S20 bound f[3H]Met-tRNA to a similar extent as the total proteins from 30 S (TP30) or 50 S (TP50) when tested by nitrocellulose filtration, in contrast to the more acidic proteins L7/L12 and S8. (2) The proteins of the peptidyltransferase centre, L2 and L16, showed no distinct specificity, binding various charged tRNAs from E. coli and Saccharomyces cerevisiae (S. cerevisiae). (3) A number of isolated ribosomal proteins hydrolysed aminoacyl-tRNA as assessed by trichloroacetic acid precipitation, in contrast to the TP30 and TP50. (4) The loss of radiolabel from Ac[14C]Phe-tRNA and from [14C]tRNA in the presence of these proteins could not be prevented by RNasin, a ribonuclease inhibitor, whereas that mediated by a sample of non-RNase-free bovine serum albumin was inhibited. (5) When double-labelled, Ac[3H]Phe-[14C]tRNA was incubated with L2 both radiolabels were lost, indicating that this potential candidate for a peptidyltransferase enzyme does not specifically cleave the ester bond between the aminoacyl residue and the tRNA.
A full-length cDNA encoding bovine ribosomal protein L3 was isolated and sequenced. The deduced protein sequence comprises 403 amino acids and shows a high level of identity with the other known mammalian L3 proteins. Southern blot analysis of bovine genomic DNA suggests that the bovine genome contains at least 4 copies of the L3 gene. A single hybridisation band of about 1.3 kb is detectable by Northern blot analysis. Within the amino acid sequence, two potential nuclear targeting sequences were detected: one at the N-terminal end and the other, consisting in a bipartite motif (amino acids 341 to 358), present and not previously noticed also in the other known mammalian L3 proteins. A search on all the available mammalian ribosomal proteins revealed the presence of this bipartite motif in many of these proteins.
Strains containing a series of restrictive and non-restrictive mutations in ribosomal protein S12 have been transformed with plasmids carrying the rrnB operon with mutations at positions 1409 and 1491 in 16S rRNA. The effects of the double-mutant constructs have been measured by growth rate, paromomycin and streptomycin sensitivity, resistance and dependence. The results demonstrate a functional interaction between the 1409-1491 region of rRNA and ribosomal protein S12.
The sequence analysis of the L1 ribosomal protein (r-protein) gene of Xenopus laevis has revealed a strong homology in four out of the nine introns of the gene; this homology region spans 60 nucleotides (nt) with 80% homology [Loreni et al., EMBO J. 4 (1985) 3483-3488]. We have extended our analysis to X. tropicalis, a species which is closely related to X. laevis. Partial sequencing of the isolated L1 gene has revealed that these 60-nt homology regions are also present in at least two introns of the X. tropicalis L1 gene. Computer analysis has revealed that perfect nt sequence complementarity exists between 13 nt of this intron region and the 28S ribosomal RNA in a region which is conserved in all eukaryotes, suggesting a possible base-pairing interaction between these two sequences.
Ribosomal proteins from Mycobacterium tuberculosis H37 Rv were analyzed by polyacrylamide gel electrophoresis and 73 proteins were separated. The 30S subunit consists of 26 proteins, the 50S subunit of 41 proteins, 6 proteins of the ribosomes could not be shown in the subunits. The molecular weights for the proteins of the 30S subunit are 9100-43500, and for the proteins of the 50S subunit 8000-46000. The sedimentation coefficient have values of 28.8S and 47.7S. It was demonstrated that streptomycin-resistant cells in comparison to sensitive cells have two additional 30S proteins. Moreover, the protein L34 has changed its position. Kanamycin- and capreomycin-resistant cells have also two additional proteins, but viomycin shows no changes.