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PubMed1980

Ribosome-membrane interactions: characterization of ribosomal proteins from loose and tight bound ribosomes.

Membrane-bound ribosomes were isolated from a post-mitochondrial supernatant fraction of mouse liver homogenate by sedimentation in a sucrose density gradient, Loose ribosomes were released from the membrane fragments with 0.5 M KCl, while tight bound ribosomes were not released. After purification of the loose and tight ribosomes subclasses, ribosomal subunit proteins were isolated and compared by two-dimensional polyacrylamide gel electrophoresis. No differences in the ribosomal protein composition was detected.

Animals↗
PubMedDate not supplied

Proteolytic degradation of ribosomal proteins during isolation of ribosomes and ribosomal subunits from Tetrahymena.

A preparation procedure previously used to isolate active ribosomal subunits from an amicronucleate strain of Tetrahymena of undefined phenoset (T. "pyriformis" CGL) yields inactive subunits when applied to other amicronucleate or to micronucleate strains of this protozoa. Proteolytic degradation of a small number of ribosomal proteins during preparation of ribosomal subunits from these strains explains this results. If cell extraction and ribosome isolation are carried out in the presence of iodoacetamide, proteolytic activity is inhibited and active ribosomal subunits are obtained. Comparison of the protein complements of active ribosomal subunits prepared in the presence of iodoacetamide from three amicronucleate strains of Tetrahymena reveals small but significant differences.

Animals↗
PubMed1976

Demonstration of ribosome-dependent photoinduced chain breakage of the 16S ribosomal ribonucleic acid component of the Escherichia coli 30S ribosomal subunit.

The effects of 253.7-nm radiation on the structural integrities of free and ribosome-bound 16S ribosomal ribonucleic acid (rRNA) have been elucidated. Exposure of aqueous solutions of Escherichia coli 30S ribosomal subunits to 253.7-nm radiation results in RNA-chain scission and the formation of single-stranded breaks in double-stranded regions of the ribosome-bound 16S rRNA. The minimum doses of incident 253.7-nm radiation required for the first detection of the two types of RNA chain breaks are 2 X 10(20) quanta for single-strand breaks in double-stranded regions of the ribosome-bound 16S rRNA,and at least 5 X 10(20) quanta for RNA-chain scission. In contrast to the photosensitivity of ribosome-bound 16S rRNA toward chain breakage, free 16S rRNA seems to be resistant toward photoinduced chain breakage at doses of incident 253.7-nm radiation up to at least 10(21) quanta.

Escherichia coli↗
PubMed1993

Fusidic acid-dependent ribosomal complexes protect Escherichia coli ribosomes from the action of the type 1 ribosome-inactivating protein crotin 2.

The type 1 ribosome-inactivating protein crotin 2 depurinated Escherichia coli ribosomes which, upon treatment of the isolated rRNA with acid aniline, released a fragment of around 240 nucleotides whose 5'-end sequence was 5'-GAGGACCGGAGUGGAC-3'. The formation of fusidic acid-dependent ribosomal complexes completely prevented release of the fragment. Ribosomes from crotin 2-pretreated fusidic acid complexes were insensitive to acid aniline. They released the RNA fragment only after a second treatment with crotin 2 and acid aniline whereas unprotected ribosomes released the fragment directly after acid aniline.

Apurinic Acid↗
PubMed1975

Membrane-bound ribosomes of myeloma cells. I. Preparation of free and membrane-bound ribosomal fractions. Assessment of the methods and properties of the ribosomes.

A cell fractionation procedure is described which allowed, by use of MOPC 21 (P3K) mouse plasmocytoma cells in culture, the separation of the cytoplasmic free and membrane-bound ribosomes in fractions devoid of mutual cross-contamination, and in which the polyribosomal structure was entirely preserved. This was achieved by sedimentation on a discontinuous sucrose density gradient in which the two ribosome populations migrate in opposite directions. A variety of controls (electron microscopy, labeling of membrane lipids, further repurification of the isolated fractions) provided no evidence of cross-contamination of these populations. However, when an excess of free 60S or 40S subunits, labeled with a different isotope, was added to the cytoplasmic extract before fractionation, the possibility of a small amount of trapping and/or adsorption of free ribosomal particles by the membrane fraction was detected, especially in the case of the 60S subunits; this could be entirely prevented by the use of sucrose gradients containing 0.15 M KC1. EDTA treatment of the membrane fraction detached almost all the 40S subunits, and about 70% of the 60S subunits. 0.5 M KC1 detached only 10% of the ribosomal particles, which consist of the native 60S subunits and the monoribosomes, i.e. the bound particles inactive in protein synthesis. Analysis in CsC1 buoyant density gradients of the free and membrane-bound polyribosomes and of their derived 60S and 40S ribosomal subunits showed that the free and membrane-bound ribosomal particles have similar densities.

Cell Fractionation↗
PubMed1985

Synthesis and incorporation of human ribosomal protein S14 into functional ribosomes in human-Chinese hamster cell hybrids containing human chromosome 5: human RPS14 gene is the structural gene for ribosomal protein S14.

In Chinese hamster ovary cells, mutations in the RPS14 gene (which was previously designated emtB) render cells resistant to normally cytotoxic concentrations of the protein synthesis inhibitor, emetine. Several lines of evidence indicate the RPS14 gene in Chinese hamster is the structural gene for ribosomal protein S14, including the finding that mutants with alterations in this gene produce an electrophoretically altered form of this protein. A human gene which complements the defect in CHO RPS14 mutants and renders them sensitive to emetine has previously been assigned to the long arm of chromosome 5. The analysis of ribosomal proteins extracted from CHO Emtr X human cell hybrids, which contain human chromosome 5 and are emetine sensitive, demonstrated the presence of both the normal human and altered hamster forms of ribosomal protein S14. Human chromosome 5, the emetine-sensitive phenotype, and the human form of ribosomal protein S14 segregate concordantly from hybrids, confirming that the human gene in question is the structural gene for this protein. In addition, the results indicate that in interspecific cell hybrids, the human form of S14 is either incorporated into functional ribosomes more efficiently than the altered hamster protein or the human gene is overexpressed relative to the corresponding hamster gene.

Animals↗
PubMed2005

Ribosomes containing mutants of L4 ribosomal protein from Thermus thermophilus display multiple defects in ribosomal functions and sensitivity against erythromycin.

Protein L4 from Thermus thermophilus (TthL4) was heterologously overproduced in Escherichia coli cells. To study the implication of the extended loop of TthL4 in the exit-tunnel and peptidyltransferase functions, the highly conserved E56 was replaced by D or Q, while the semiconserved G55 was changed to E or S. Moreover, the sequence -G55E56- was inverted to -E55G56-. When we incorporated these mutants into E. coli ribosomes and investigated their impact on poly(Phe) synthesis, high variations in the synthetic activity and response to erythromycin of the resulting ribosomes were observed. In the absence of erythromycin, ribosomes harboring mutations G55E and E56D in TthL4 protein were characterized by low activity in synthesizing poly(Phe) and decreased capability in binding tRNA at the A site. On the other hand, ribosomes possessing mutations G55E, G55S, G55E-E56G, or E56Q in TthL4 protein were unexpectedly more sensitive to erythromycin. Evidence in support of these findings was drawn by in vivo experiments, assessing the erythromycin sensitivity of E. coli cells expressing wild-type or mutant TthL4 proteins. Our results emphasize the role of the extended loop of L4 ribosomal protein in the exit-tunnel and peptidyltransferase center functions.

Drug Resistance, Microbial↗
PubMed1987

The primary structure of rat ribosomal protein S12. The relationship of rat S12 to other ribosomal proteins and a correlation of the amino acid sequences of rat and yeast ribosomal proteins.

The covalent structure of the rat 40 S ribosomal subunit protein S12 was determined from the sequence of amino acids in tryptic, chymotryptic, thermolytic, and cyanogen bromide peptides and inferred from the sequence of nucleotides in a recombinant cDNA. Rat ribosomal protein S12 contains 129 amino acids and has a molecular weight of 14,120. The amino acid sequences of a number of ribosomal proteins appear to be related to rat S12. These include spinach chloroplast L7, Escherichia coli S5, Nicotiana tabacum chloroplast S18, and Bacillus stearothermophilus S12, and perhaps others. In addition, there are two sequences, 26 and 18 amino acids in length, in rat S12 that may be related to segments of the same number of residues in ribosomal proteins from a number of species. These, and other results, reinforce the suggestion that ribosomal proteins form an extended family.

Amino Acid Sequence↗
PubMed1993

The primary structure of rat ribosomal protein L23a. The application of homology search to the identification of genes for mammalian and yeast ribosomal proteins and a correlation of rat and yeast ribosomal proteins.

The amino acid sequence of the rat 60 S ribosomal subunit protein L23a was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L23a has 156 amino acids and a molecular weight of 17,684. Hybridization of the L23a cDNA to digests of nuclear DNA suggests that there are 18-20 copies of the L23a gene. The mRNA for the protein is about 600 nucleotides in length. Rat L23a is related to the yeast Saccharomyces cerevisiae L25, to the archaebacterial Methanococcus vannielii L23, to eubacterial Escherichia coli L23, and to other members of the L23 family of ribosomal proteins. A novel application of a routine homology search procedure was employed to identify a nucleotide sequence that could be used to design an oligodeoxynucleotide probe to screen a library for a cDNA that encodes rat L23a; this same procedure uncovered a number of previously unidentified genes for yeast ribosomal proteins in the GenBank DNA data base. In a correlation of rat and yeast ribosomal proteins 48 pairs are shown to be related.

Amino Acid Sequence↗
PubMed1993

Derivatives of the yeast mitochondrial ribosomal protein MrpS28 replace ribosomal protein S15 as functional components of the Escherichia coli ribosome.

The mitochondrial ribosomal protein MrpS28 is considerably larger than its eubacterial homolog, Escherichia coli ribosomal protein S15 (Eco S15). Relative to a region of homology that spans the entire length of the bacterial protein, mature MrpS28 is extended by 117 and 48 amino acids at its amino and carboxyl termini, respectively. Both the amino-terminal and S15-like domains of MrpS28 are essential for function in yeast mitochondria. Here, we show that these same two domains function in E. coli. The S15-like domain of MrpS28 alone complements a cold-sensitive mutation in E. coli strain KR121 that gives rise to reduced levels of Eco S15. However, complementation by the S15-like domain of MrpS28 is inefficient when compared with Eco S15. Surprisingly, the amino-terminal domain of MrpS28, which is apparently a unique component of the mitochondrial ribosome and is unable by itself to complement the cold-sensitive phenotype, enhances the ability of the S15-like domain to support growth of KR121 cells at nonpermissive temperatures. Together, these data suggest that the amino-terminal domain contributes to the fundamental properties of MrpS28 involved in the assembly and function of both mitochondrial and E. coli ribosomes.

Amino Acid Sequence↗
PubMed1984

The synthesis of ribosomal RNA and ribosomal protein and their incorporation into ribosomes in the uterus of the oestrogen-stimulated immature rat.

The effect of oestrogen on the synthesis of ribosomal proteins in the uterus of the immature rat has been investigated. Stimulated synthesis peaks, at 6-7-times control levels, 12 h after a single administration of the hormone. The stimulated synthesis and incorporation of newly made proteins into ribosomal particles exhibit very similar kinetics. The incorporation of newly made rRNA into ribosomes mirrors that of ribosomal protein but lags several hours behind the peak of oestrogen-stimulated rRNA synthesis.

Animals↗
PubMed1970

Dissociation of 70 S E. coli ribosomes induced by a ribosomal factor (DF). Electrophoretic studies of the ribosomal particles.

The dissociation of purified 70 S.E. coli ribosomes, induced by the dissociation factor DF, has been studied by submitting the reaction mixtures to electrophoresis on polyacrylamide gels. The electrophoretic analysis of the ribosome mixtures revealed a heterogeneity which escaped detection by conventional sucrose gradient centrifugation. Increasing amounts of DF in the reaction mixtures converted 70 S ribosomes to particles (designated 70 S (I)) which migrate slower in the electric field than the original 70 S ribosomes. These 70 S (I) ribosomes still consist of both subunits. They dissociate upon further raising the DF concentration.

Journal Article↗
PubMed1975

Membrane-bound ribosomes of myeloma cells. II. Kinetic studies on the entry of newly made ribosomal subunits into the free and the membrane-bound ribosomal particles.

The kinetics of appearance of newly made 60S and 40S ribosomal subunits in the free and membrane-bound ribosomal particles of P3K cells were explored by determining the specific radioactivities of their 18S and 28S RNA after various lengths of [3H]uridine pulse. Both 40S and 60S subunits enter free and membrane-bound polyribosomes at comparable rates from the cytoplasmic pool of newly made, free native subunits, the 40S subunits entering the native subunit pool and the polyribosomes slightly earlier than the 60S subunits. At all times, the specific radioactivity of the membrane-bound native 60S subunits was slightly lower than that of the polyribosomal 60S subunits. This indicates that the membrane-bound native 60S subunits are not precursors destined to enter membrane-bound polyribosomes and suggests that they result from the dissociation of ribosomes after chain termination. The results observed also suggest that the membrane-bound native 60S subunits are not reutilized before their release from the membranes, which probably takes place shortly after dissociation from their 40S subunits. The monoribosomes, both free and membrane-bound, had the lowest specific radioactivities in their subunits. Finally, a small amount of newly made native 40S subunits, containing 18S RNA of high specific radioactivity, and apparently also newly made messenger RNA were detected on the membranes. The high turnover of these membrane-bound native 40S subunits suggests that they may represent initiation complexes formed with mRNA which has just reached the membranes and which has not yet given rise to polyribosomes.

Cell Line↗
PubMed1990

The ribosomal domain of the bacterial release factors. The carboxyl-terminal domain of the dimer of Escherichia coli ribosomal protein L7/L12 located in the body of the ribosome is important for release factor interaction.

1. Polyclonal antibodies (pAb 1-73 and pAb 26-120) have been raised against both an N-terminal fragment of Escherichia coli ribosomal protein L7/L12 (amino acids 1-73), and a fragment lacking part of the N-terminal domain (amino acids 26-120). 2. Only pAb 26-120 inhibited release-factor-dependent in vitro termination functions on the ribosome. This antibody binds over the length of the stalk of the large subunit of the ribosome as determined by immune electron microscopy, thereby not distinguishing between the C-terminal domains of the two L7/L12 dimers, those in the stalk or those in the body of the subunit. 3. A monoclonal antibody against an epitope of the C-terminal two thirds of the protein (mAb 74-120), which binds both to the distal tip of the stalk as well as to a region at its base, reflecting the positions of the two dimers is strongly inhibitory of release factor function. 4. A monoclonal antibody against an epitope of the N-terminal fragment of L7/L12 (mAb 1-73), previously shown to remove the dimer of L7/L12 in the 50S subunit stalk but still bind to the body of the particle, partially inhibited release-factor-mediated events. 5. The mAb 74-120 inhibited in vitro termination with a similar profile when the stalk dimer of L7/L12 was removed with mAb 1-73, indicating that the body L7/L12 dimer, and in particular its C-terminal domains, are important for release factor/ribosome interaction. 6. The two release factors have subtle differences in their binding domains with respect to L7/L12.

Amino Acids↗
PubMed2000

Mutations at position A960 of E. coli 23 S ribosomal RNA influence the structure of 5 S ribosomal RNA and the peptidyltransferase region of 23 S ribosomal RNA.

The proximity of loop D of 5 S rRNA to two regions of 23 S rRNA, domain II involved in translocation and domain V involved in peptide bond formation, is known from previous cross-linking experiments. Here, we have used site-directed mutagenesis and chemical probing to further define these contacts and possible sites of communication between 5 S and 23 S rRNA. Three different mutants were constructed at position A960, a highly conserved nucleotide in domain II previously crosslinked to 5 S rRNA, and the mutant rRNAs were expressed from plasmids as homogeneous populations of ribosomes in Escherichia coli deficient in all seven chromosomal copies of the rRNA operon. Mutations A960U, A960G and, particularly, A960C caused structural rearrangements in the loop D of 5 S rRNA and in the peptidyltransferase region of domain V, as well as in the 960 loop itself. These observations support the proposal that loop D of 5 S rRNA participates in signal transmission between the ribosome centers responsible for peptide bond formation and translocation.

Aldehydes↗
PubMed1990

Localization of an oligodeoxynucleotide complementing 16S ribosomal RNA residues 520-531 on the small subunit of Escherichia coli ribosomes: electron microscopy of ribosome-cDNA-antibody complexes.

The oligodeoxynucleotide dACCGCGGCTGCT, complementary to Escherichia coli small ribosomal subunit RNA residues 520-531, has been used to probe subunit conformation and to localize the sequence in the subunit. Conditions for binding of the cDNA to 30S subunits were optimized and specificity of the interaction was demonstrated by RNase H cleavage. Three kinds of terminal modification of this cDNA were used to allow its localization by immune electron microscopy. A solid phase support with 5'-dimethoxytrity-N6-delta 2-isopentenyl-adenosine linked to controlled pore glass was synthesized, and used to prepare oligomer with an added 3'-terminal residue of isopentenyl adenosine. cDNA with a 5' primary amine substituent was modified with 1-fluoro-2,4-dinitrobenzene to prepare 5'-dinitrophenyl oligonucleotide, and both modifications together gave doubly-derivatized probes. Immune electron microscopy with antibodies to dinitrophenol, isopentenyl adenosine, or both, was used to place the cDNA on 30S subunits. In each case the probe was placed at a single site at the junction of the head and body of the subunit, near the decoding site and the area in which elongation factor Tu is bound. It is proposed that this segment of ribosomal RNA functions in mRNA binding and orientation.

Antibodies↗
PubMed2004

The PRC-barrel domain of the ribosome maturation protein RimM mediates binding to ribosomal protein S19 in the 30S ribosomal subunits.

The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes. A DeltarimM mutant is defective in 30S maturation and accumulates 17S rRNA. To study the interaction of RimM with the 30S and its involvement in 30S maturation, RimM amino acid substitution mutants were constructed. A mutant RimM (RimM-YY-->AA), containing alanine substitutions for two adjacent tyrosines within the PRC beta-barrel domain, showed a reduced binding to 30S and an accumulation of 17S rRNA compared to wild-type RimM. The (RimM-YY-->AA) and DeltarimM mutants had significantly lower amounts of polysomes and also reduced levels of 30S relative to 50S compared to a wild-type strain. A mutation in rpsS, which encodes r-protein S19, suppressed the polysome- and 16S rRNA processing deficiencies of the RimM-YY-->AA but not that of the DeltarimM mutant. A mutation in rpsM, which encodes r-protein S13, suppressed the polysome deficiency of both rimM mutants. Suppressor mutations, found in either helices 31 or 33b of 16S rRNA, improved growth of both the RimM-YY-->AA and DeltarimM mutants. However, they suppressed the 16S rRNA processing deficiency of the RimM-YY-->AA mutant more efficiently than that of the DeltarimM mutant. Helices 31 and 33b are known to interact with S13 and S19, respectively, and S13 is known to interact with S19. A GST-RimM but not a GST-RimM(YY-->AA) protein bound strongly to S19 in 30S. Thus, RimM likely facilitates maturation of the region of the head of 30S that contains S13 and S19 as well as helices 31 and 33b.

Alanine↗