Showing papers on "Ribosomal protein published in 1979"

The structure and function of eukaryotic ribosomes.

Abstract: PERSPECTIVES AND SUMMARY . GENERAL CHARACTERISTICS OF EUKARYOTIC RIBOSOMES .. ISOLATION AND CHARACTERIZATION OF EUKARYOTIC RIBOSOMAL PROTEINS .. Purification . Characterization . The Number of Ribosomal Proteins .. Stoichiometry . Sequence Analyses . THE ASSOCIATION OF RIBOSOMAL PROTEINS AND RIBOSOMAL RNA PHOSPHORYLATION OF EUKARYOTIC RIBOSOMAL PROTEINS .. Structure of S6 . Conservation of S6 . Conditions Altering the Phosphorylation of S6 . Acidic Ribosomal Phosphoproteins .. Relation of Rat Liver PI and P2 to E. Coli L7/L12 .. Artemia Salina Acidic Ribosomal Phosphoproteins ..

Nucleotide sequence of the ribosomal protein gene cluster adjacent to the gene for RNA polymerase subunit beta in Escherichia coli

Abstract: The 3072-nucleotide-long sequence of a segment from the 88-min region of the Escherichia coli chromosome has been determined. The sequence covers the genes for ribosomal proteins L11 (rplK), LI (rplA), L10 (rplJ), and L7/L12 ((rplL), and the 5' end of the gene for the beta subunit of RNA polymerase (rpoB), along with the presumed regulatory regions for these genes. The probable locations of the promoter for the first two genes (the L11 operon) and the promoter for the latter three genes (the proximal part of the beta operon) have been identified. We have also found that the four ribosomal protein genes preferentially use codons that are recognized efficiently by the most abundant tRNA species. These and other features of the sequence results are discussed in relation to available information obtained from both in vitro and in vivo experiments on the expression of these ribosomal and RNA polymerase subunit genes.

Phenotypic suppression and misreading in Saccharomyces cerevisiae

Abstract: A NUMBER of antibiotics and other inhibitors have been useful in genetic and biochemical analyses of the protein-synthesising machinery of prokaryotic and eukaryotic organisms. Aminoglycoside antibiotics have been shown to be particularly helpful in this respect, especially in identifying ribosomal protein cistrons in bacteria. The aminoglycosides cause extensive misreading of the RNA code words in vitro1 and suppress many nonsense and missense mutations in E. coli2 phenotypically. The misreading observed in cell-free translation is believed to be the basis for the phenotypic suppression, although the exact mechanism is not known. Apart from a report of suppression of a single mutation in yeast by streptomycin3, there have been no demonstrations of phenotypic suppression in eukaryotic organisms. Earlier studies of mistranslation in vitro in eukaryotic systems indicated that this phenomenon is rare. Streptomycin did not cause misreading with cytoplasmic ribosomes of rat liver4, rabbit spleen5, chicken liver6 or yeast7; neomycin had no effect in rabbit reticulocyte extracts4 and only a slight effect in the yeast and chicken liver systems6,7. These results suggested that translation in higher organisms functions with higher fidelity than that in bacteria. Nevertheless, aminoglycoside antibiotics have recently been shown to cause extensive translational misreading in vitro in systems derived from Tetrahymena8, wheat embryo9, and cultured human cells10.

Isolation of a 7S particle from Xenopus laevis oocytes: a 5S RNA-protein complex.

Abstract: Previtellogenic oocytes of Xenopus laevis contain a free 5S RNA-protein complex sedimentating at 7 S. This particle consists of one molecule of 5S RNA and one 45,000-dalton protein. The protein of the 7S particle and the protein that is released in association with 5S RNA when the ribosome is treated with EDTA are unrelated. Because the 5S RNA accumulated by small oocytes in storage particles is incorporated into the ribosome later in oogenesis, we conclude that 5S RNA is succesively associated with two proteins during the life span of the oocyte.

Preparation and analysis of mitochondrial ribosomes.

Abstract: Publisher Summary The chapter discusses the preparation and analysis of mitochondrial ribosomes. Mitochondria contain a distinct species of ribosome that functions in the synthesis of specific polypeptides of the inner mitochondrial membrane. The mitochondrial (mit) ribosomes of microorganisms and higher plants have sedimentation coefficients (s) of 70-80 S. Distinctive features of mit ribosomes include their sensitivity to inhibitors of bacterial protein synthesis, dissociation of monomers at relatively high Mg 2+ concentrations. Mit ribosomes are isolated from many organisms by procedures that include the following steps: (a) isolation of mitochondria, (b) lysis of mitochondria using either deoxycholate or a nonionic detergent, (c) preparation of a mit ribosomal pellet, and (d) separation of monomers or subunits on sucrose gradients. The chapter emphasizes two features: purification of the mitochondria by flotation gradient centrifugation and substitution of Ca 2+ for Mg 2+ during mitochondrial lysis to inhibit nuclease activity. A number of conventional electrophoretic systems are adapted for the analysis of mit ribosomal proteins.

Proposed uniform nomenclature for mammalian ribosomal proteins.

Abstract: The numbering systems for mammalian ribosomal proteins used in several laboratories have been correlated and a proposal for a standard system is presented.

Multiple phosphorylation of ribosomal protein S6 during transition of quiescent 3T3 cells into early G1, and cellular compartmentalization of the phosphate donor.

Abstract: At 5 min after quiescent cells are induced to enter G1 there is a large increase in the amount of 32P incorporated into 40S ribosomal protein S6. Here we show that changes in the specific activities of 32Pi and [gamma-32P]ATP in stimulated as compared to quiescent cultures do not account for this large increase. Instead, we demonstrate by decreased electrophoretic mobility on two-dimensional polyacrylamide gels that this increase is due to a quantitative increase in the total amount of phosphate incorporated into S6. Furthermore, pulse-chase experiments show that the phosphate that is incorporated into S6 is metabolically stable during at least the first 60 min of induction and that the incorporation of 32P into S6 responds immediately to the replacement of 32Pi by Pi in the medium, in contrast to [gamma-32P]ATP which changes very slowly. Thus, the S6 phosphate donor must be a compartment separate from that of the total cellular ATP.

Acidic ribosomal proteins from eukaryotic cells. Effect on ribosomal functions.

Abstract: Precipitation of Saccharomyces cerevisiae ribosomes by ethanol under experimental conditions that do not release the ribosomal proteins can affect the activity of the particles. In the presence of 0.4 M NH4Cl and 50% ethanol only the most acidic proteins from yeast and rat liver ribosomes are released. At 1 M NH4Cl two more non-acidic proteins are lost from the ribosomes. The release of the acidic proteins causes a small inactivation of the polymerizing activity of the particles, additional to that caused by the precipitation itself. The elongation-factor-2-dependent GTP hydrolysis of the ribosomes is, however, more affected by the loss of acidic proteins. These proteins can stimulate the GTPase but not the polymerising activity when added back to the treated particles. Eukaryotic proteins cannot be sustituted for bacterial acidic proteins L7 and L12. We have not detected immunological cross-reaction between acidic proteins from Escherichia coli and those from yeast, Artemia salina and rat liver or between acidic proteins from these eukaryotic ribosomes among themselves.

Regulation of ribosomal protein synthesis in Escherichia coli by selective mRNA inactivation.

Abstract: In an Escherichia coli strain lysogenic for lambda spc2 transducing phage, an extra copy of ribosomal protein (r-protein) genes in the spc and alpha operons are carried on the phage chromosome. Expression of genes in the spc operon in this merodiploid strain was compared with that in a control "haploid" strain carrying lambda trkA phage. It was found that the synthesis rate of spc mRNA, relative to other reference mRNA in the merodiploid strain, is about 2-fold higher than that in the control strain; yet, no dosage effect was observed in the synthesis rate of r-proteins in the spc or alpha operon. The spc mRNA was found to be more rapidly degraded in the merodiploid strain than in the control strain, and its steady-state amount, relative to reference mRNA, was only slightly higher in the merodiploid strain than in the control strain. Thus, E. coli cells have the ability to regulate the rate of r-protein synthesis regardless of the rate of transcription of r-protein genes, presumably by inactivation of the mRNA followed by its degradation. A model is proposed which involves selective inactivation of r-protein mRNA by a feedback mechanism. The model can explain coordinated synthesis of r-proteins and other observations related to selective expression of certain alleles in diploid strains.

Spot position of rat liver ribosomal proteins by four different two-dimensional electrophoreses in polyacrylamide gel.

Abstract: Separation of the proteins from rat liver 40S and 60S ribosomal subunits and polysomes was done in four different two-dimensional polyacrylamide gel electrophoresis systems. The first dimension was run at acidic or basic pH, the second dimension either with sodium dodecyl sulphate or at acidic pH in 18% acrylamide. The position of each individual protein of both subunits and polysomes was determined in each system. This identification resulted from a new method avoiding any previous purification of individual proteins. The new “proposed uniform nomenclature for mammalian ribosomal proteins” (McConkey et al. in press) was used for numbering the proteins in the four systems.

Selection for Escherichia coli mutants with proteins missing from the ribosome.

Abstract: Antibiotic-independent revertants of an erythromycin-dependent strain of Escherichia coli were isolated by spontaneous selection. Their ribosomal proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. In contrast to most ribosomally targeted selections, the specific absence of a certain protein from the ribosome, rather than alterations in ribosomal proteins, was observed. Mutants were found with protein S20, L11, L15, L28, L29, or L30 missing.

Binding of Thiostrepton to a Complex of 23‐S rRNA with Ribosomal Protein L11

Abstract: Thiostrepton binds with high affinity and with a 1:1 stoichiometry to a complex formed between Escherichia coli 23-S ribosomal RNA and ribosomal protein L11 of E. coli or the homologous protein BM-L11 of Bacillus megaterium. In the presence of T1 ribonuclease, protein BM-L11 and thiostrepton protect from degradation a fragment of E. coli 23-S RNA estimated to be about 50 nucleotides in length.

[40] Purification of ribosomal proteins from Escherichia coli under nondenaturing conditions

Abstract: Publisher Summary The chapter discusses the purification of ribosomal proteins from Escherichia coli under nondenaturing conditions. These methods are developed to isolate proteins from the Escherichia coli ribosome that are in a more native state than those that are previously isolated in the presence of acetic acid and urea. The term “native” does not necessarily imply that the purified proteins are in the same conformational state as they are to be found in Situ on the ribosome because under these conditions protein-protein and protein-RNA interactions certainly play an important part. The conditions used for this purification procedure do not involve protein denaturants such as urea and extreme pH or lyophilization, but employ a high-salt extraction with LiCl followed by fractionation in the presence of salt. High salt concentrations especially of LiC1 are known to perturb the tertiary structure of proteins. The salt-extracted proteins are more soluble at high ionic strength and less soluble at low salt concentrations. This is the reverse of the solubility exhibited by previously prepared ribosomal protein.

Protein Synthesis in Imbibing Wheat Embryos

Abstract: Polypeptides synthesized by imbibing wheat embryos have been compared with those made by cell-free extracts programmed with bulk poly(A)-rich RNA from dry wheat embryos. Newly synthesized polypeptides, labeled with [35S]methionine, were resolved by one-dimensional and two-dimensional electrophoresis and then records of the separations were prepared by fluorography. When programmed by bulk poly(A)-rich RNA from dry wheat embryos, a nuclease-treated rabbit reticulocyte lysate synthesizes an array of polypeptides which is broadly similar to that formed when a wheat germ extract is programmed with the same RNA. Polypeptides made in both homologous and heterologous cell-free systems, under the direction of bulk poly(A)-rich RNA from dry wheat embryos, are broadly similar to those formed during early (0--40 min) imbibition of dry wheat embryos. As imbibition progresses beyond 40 min, there are profound changes in the one-dimensional and two-dimensional electrophoretic distributions of newly made polypeptides present in the 23 000 x g supernatant fraction of cell-free homogenates; characteristically, low-molecular-weight and basic polypeptides comprise a diminishing proportion of the total polypeptides as imbibition progresses beyond 40 min. Ribosomal proteins are conspicuous among the proteins formed during early imbibition and especially prominent among the products formed when homologous cell-free polypeptide synthesis is programmed by bulk poly(A)-rich RNA from dry wheat embryos.

Expression of Escherichia coli ribosomal protein and RNA polymerase genes cloned on plasmids.

Abstract: Fragments of λdrif d 18 DNA with different end-points within the set of structural genes of ribosomal proteins L11 (rplK), L1 (rplA), L10 (rplJ) and L12 (rplL) as well as the β (rpoB) and β′ (rpoC) subunits of RNA polymerase have been cloned on plasmids. These plasmids were transformed in host cells which were mutant for each of the genes, enabling expression of both wild-type (plasmid-borne) and mutant (chromosomal) genes to be differentiated. On the basis of these results we propose the following genetic structure for the region: rplK and rplA are in one operon; rplL, rpoB and rpoC are in a second. Our data suggest the possibility that rplJ is by itself in an operon situated between the other two.

Proteins of small subunits of rat liver ribosomes that interact with poly(U). II. Cross-links between poly(U) and ribosomal proteins in 40 S subunits induced by UV irradiation.

Abstract: (1) When rat liver 40 S ribosomal proteins in 6 M urea were were mixed with poly(U) at an appropriate ratio, a precipitate was formed which was also insoluble in the sample solution for two-dimensional acrylamide gel electrophoresis. Analyses by two-dimensional acrylamide gel electrophoresis showed that S7 and S10 proteins (according to our numbering system) had disappeared selectively from the fraction soluble in 6 M urea. These two proteins were present in the fraction insoluble in 6 M urea, and became soluble in the sample solution after treating it with RNase. The results suggest that S7 and S10 proteins have strong affinities for poly(U). When rat liver 40 S subunits were incubated with poly(U), similar results were obtained. (2) After incubation of 40 S subunits with [3H]poly(U) and then with unlabeled poly(U), UV irradiation cross-linked poly(U) to the protein moiety of the 40 S subunit. When the protein fraction insoluble in the sample solution for two-dimensional electrophoresis was prepared from 40 S subunits cross-linked to poly(U) and then subjected to two-dimensional acrylamide gel electrophoresis after RNase treatment, S7 and S10 proteins were detected on the gel. In addition to the S7 protein spot, a triangular area spreading from the spot to the origin contained radioactivity. The results suggest that poly(U) is cross-linked to S7 protein and oligo(U) fragments bound to S7 protein affect its electrophoretic mobility. (3) Ribosomal proteins were prepared from 40 S subunits cross-linked to carrier-free [3H]poly(U) and analyzed by three-dimensional acrylamide gel electrophoresis (Terao, K. & Ogata, K. (1975) Biochim. Biophys. Acta 402, 214--229) after RNase treatment. It was found that S7, S6, and S15 proteins are cross-linked to poly(U). From the results of the present and preceding experiments it is concluded that S7 is the poly(U)-binding protein. The possibility that other proteins in 40 S ribosomal subunits interact with poly(U) is discussed.

Total reconstitution of 50 S subunits from Escherichia coli ribosomes.

Abstract: Publisher Summary The 50 S subunit from E. coli ribosomes consists of 34 proteins and two RNA molecules and the 30 S subunit consists of 21 proteins and 16 S RNA. In spite of the large number of ribosomal components, which are all present in one copy per ribosome 2 (except L7/L12:3 or 4 copies), the subunits can be self-assembled in vitro by a technique known as “total reconstitution.” The total reconstitution of the large subunit from E. coli ribosomes requires a two-step incubation of the components (44°, 4 mM Mg 2+ → 50°, 20 mM Mg 2+ ). A conformational change in reconstituted intermediates is the rate-limiting step, and these conformational changes have different ionic and incubation optima. In addition to its importance for assembly studies, the technique of reconstitution is used successfully for the elucidation of the functional role of a number of ribosomal components. The two-step reconstitution leads to particles that are structurally indistinguishable from native 50 S subunits and show 50-100% of the activity of native subunits in various functional tests.

Operon-specific regulation of ribosomal protein synthesis in Escherichia coli.

Abstract: We have cloned a DNA fragment harboring the genes for ribosomal proteins L2, L4, and L23 on a plasmid vector that contains a lac operator and promoter. The cloned ribosomal protein genes are now under the control of lacOP. Addition of a lac inducer to these cells results in a specific 5- to 10-fold increase in the synthesis of the proteins corresponding to the cloned genes. Within 10 min of this induction, the synthesis of ribosomal proteins S3, S19, L3, L16, L22, and L29 stops almost completely. The genes for all these proteins reside in the same chromosomal operon as L2, L4, and L23. We have seen no dramatic effect on the synthesis of any other ribosomal proteins. Thus, the induction of L2, L4, and L23 results in a specific and rapid decrease in the expression of all (or almost all) genes in their own transcription unit.

Peptide chain elongation rate and ribosomal activity in Saccharomyces cerevisiae as a function of the growth rate.

Abstract: The peptide-chain elongation rate of Saccharomyces cerevisiae at two different growth rates was estimated by the kinetics of radioactive labelling of nascent and finished polypeptides as described by Gausing, 1972, and Young and Bremer, 1976. The elongation rates of a diploid strain cultured in yeast nitrogen base supplemented with glucose or acetate were 9.3 amino acids/s and 5.5 amino acids/s at 30°C, respectively. These data together with published values on the “ribosomal efficiency” as a function of growth rate (Waldron and Lacroute, 1975) enable us to estimate the rate of synthesis of ribosomal proteins as a function of the rate of total protein synthesis, αr, and the fraction of ribosomes that are active in protein synthesis. We conclude that in S. cerevisiaeαr, is largely independent of the growth rate while the fraction of active ribosomes decreases with decreasing growth rate.

Expression of ribosomal protein genes cloned in a hybrid plasmid in Escherichia coli: gene dosage effects on synthesis of ribosomal proteins and ribosomal protein messenger ribonucleic acid.

Abstract: Using ColE1-TnA hybrid plasmid RSF2124 as the cloning vector, we constructed a hybrid plasmid, pNO1001, which carried seven ribosomal protein (r-protein) genes in the spc operon together with their promoter. The plasmid also carried three r-protein genes which precede the spc operon, but did not carry the bacterial promoter for these genes. Expression of r-protein genes carried by pNO1001 was studied by measuring messenger ribonucleic acid and r-protein synthesis in cells carrying the plasmid. It was found that the messenger ribonucleic acid for all the promoter-distal r-protein genes was synthesized in large excess relative to messenger ribonucleic acid from other chromosomal r-protein genes which are not carried by the plasmid. However, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. The absence of large gene dosage effects on the synthesis of other distal proteins appeared to be due, at least in part, to preferential inactivation and/or degradation of the distal message which codes for these proteins; in addition, some preferential inhibition of translation of the distal message might also have been involved. Overproduced L14 and L24 were found to be degraded in recA+ strains at both 30 and 42 degrees C; in recA strains, the degradation took place at 42 degrees C but was very slow or absent at 30 degrees C. The recA strains carrying pNO1001 failed to form colonies at 30 degrees C, presumably because of overaccumulation of r-proteins. The results suggest that degradation of excess r-proteins is an important physiological process.