Ribosomal protein as substrate for a GTP-dependent protein kinase from yeast

A protein kinase specific for casein and acidic ribosomal proteins was isolated and partly characterized.It was found that the enzyme utilizes GTP and ATP as phosphoryl donors. Its affinity for ATP was considerably higher than for GTP with the km values of 7.6 × 10^-6M and 5.5 × 10^-5M, respectively.Two-dimensional acrylamide gel electrophoresis revealed the phosphorylation of the same ribosomal proteins with either of the [-³²P] nucleotides used. It was also shown that one acidic protein (S1 or S2) of 40 S and two acidic proteins (L2 and L3) of 60 S ribosomal subunits were predominantly phosphorylated in vitro. The phosphorylated proteins: L2 and L3 seem to correspond to the proteins of L7 and L12 of E. coli ribosomes. The isolated kinase phosphorylated several basic ribosomal proteins though to a lower extent than the acidic ones.     

The effect of both homologous and heterologous DNA on integration efficiencies in pneumococcal transformation

Summary A mutant of the yeast Saccharomyces cerevisiae has been isolated that is resistant to narciclasine, an inhibitor of peptide bond formation on 80S ribsomes. The mutant shows cross-resistance to a number of inhibitors of peptidyl transferase including anthelmycin, a 4-aminohexosyl cytosine antibiotic, which does not compete with narciclasine for its ribosomal binding site. The mutation is within the gene tcm1 or a closely linked gene on chromosome XV; it is expressed in the 60S ribosomal subunit. The parameters of the binding of (³H)narciclasine to ribosomes and ribosomal subunits from both wild-type and mutant strains have been calculated by ultracentrifugation. One molecule of narciclasine is bound per ribosome or per 60S ribosomal subunit, the values of the dissociation constants being 0.054 and 0.13 m respectively, for 80S and 60S particles from the wild-type cells. Ribosomes of the mutant strain have a lower affinity for narciclasine and trichodermin than ribosomes from wild-type cells. The mutation is semidominant in heterozygous diploid cells.     

Affinity labeling of specific regions of 23 S RNA by reaction of N-bromoacetyl-phenylalanyl-transfer RNA with Escherichia coli ribosomes

Reaction of the affinity-labeling reagent N-bromoacetyl-[¹⁴C]phenylalanyl-tRNA with Escherichia coli ribosomes results in covalent labeling of 23 S ribosomal RNA in addition to the previously reported labeling of ribosomal proteins. The reaction with the 23 S RNA is absolutely dependent on the presence of messenger RNA. Covalent attachment of the affinity label to 23 S RNA was demonstrated by its integrity in strongly dissociating solvents, and the conversion of the labeled material to small oligonucleotides by ribonuclease treatment. After digestion of labeled 23 S RNA with T₁ ribonuclease, the radioactivity is found mainly in two oligonucleotide fragments. These results support models in which both ribosomal RNA and ribosomal protein contribute to the structure of the region of the ribosome surrounding the peptidyl transferase center.     

Affinity labelling of rat liver ribosomal protein S26 by heptauridylate containing a 5′-terminal alkylating group

Heptauridylate bearing a radioactive alkylating [¹⁴C]-4-(N-2-chloroethyl-N-methylamino)benzylamine attached to the 5-phosphate via amide bond, was bound to ribosomes and small ribosomal subunits from rat liver which thereby were coded to bind N-acylated Phe tRNA. After completion of the alkylating reaction and subsequent hydrolysis of the phosphamide bond ribosomal proteins were isolated. Radioactivity was found covalently associated preferentially with protein S26 and, to a very small extent, with proteins S3 and S3a. The affinity labelling reaction could be abolished by (pU)₁₄ and poly(U). From the results it is concluded that ribosomal protein S26 is located at the mRNA binding site of rat liver ribosomes.     

Loosening and unfolding of E. coli 50 S ribosomal subunits: Dependence on magnesium content and temperature

Reversible change of 50 S ribosomal subunits to 40 S particles takes place in cold buffered 0.5 M NH₄Cl solutions either containing Mg⁺⁺ (up to 0.1 M), or free from Mg⁺⁺ and even supplemented with EDTA (1 mM). The 40 S particles were stable only within a definite temperature range. Heating of the samples caused completely irreversible unflding of the 40 S particles. This melting appeared to be co-operative and took place within a very narrow range of temperature, which for samples containing Mg⁺⁺ was a linear function of the log of Mg⁺⁺ concentration.The results suggest that two types of bonds maintained the compact structure of the ribosomal subunits: ionic bonds involving Mg⁺⁺ and heat-labile weak interactions between ribosomal components.     

Interaction of the intermediate filament protein vimentin with ribosomal subunits and ribosomal RNA in vitro

If in a low ionic strength extract of Triton X-100-resistant residual cell structures derived from Ehrlich ascites tumour (EAT) cells Mg²⁺ was chelated by EDTA, vimentin became associated with unfolded ribosomal subunits. The first molecular characterization of this association has shown that (1) vimentin binds to the RNA moiety of the ribosomes, (2) vimentin has a higher affinity for unfolded small ribosomal subunits or 18S rRNA than for unfolded large ribosomal subunits or 28S rRNA, (3) the limited degradation of vimentin by the vimentin-specific, Ca²⁺-activated proteinase, with the formation of a 48 Kd breakdown product, abolishes its affinity for rRNA, (4) the association products are rather sensitive to moderate concentrations of KCl and Mg²⁺, and (5) reductive alkylation of vimentin with pyridoxal-5-phosphate and NaBH₄ has no effect on the affinity of vimentin for rRNA. Actin and tubulin do not interact with EAT cell rRNA under the above ionic conditions.     

Translation elongation factor-3 (EF-3): An evolving eukaryotic ribosomal protein?

Fungi appear to be unique in their requirement for a third soluble translation elongation factor. This factor, designated elongation factor 3 (EF-3), exhibits ribosome-dependent ATPase and GTPase activities that are not intrinsic to the fungal ribosome but are nevertheless essential for translation elongation in vivo. The EF-3 polypeptide has been identified in a wide range of fungal species and the gene encoding EF-3 (YEF3) has been isolated from four fungal species (Saccharomyces cerevisiae, Candida albicans, Candida guillermondii, andPneumocystis carinii). Computer-assisted analysis of the predictedS. cerevisiae EF-3 amino acid sequence was used to identify several potential functional domains; two ATP binding/catalytic domains conserved with equivalent domains in members of the ATP-Binding Cassette (ABC) family of proteins, an aminoterminal region showing significant similarity to theE. coli S5 ribosomal protein, and regions of predicted interaction with rRNA, tRNA, and mRNA. Furthermore, EF-3 was also found to display amino acid similarity to myosin proteins whose cellular function is to provide the motive force of muscle. The identification of these regions provides clues to both the evolution and function of EF-3. The predicted functional regions are conserved among all known fungal EF-3 proteins and a recently described homologue encoded by the Chlorella virus CVK2. We propose that EF-3 may play a role in the ribosomal optimization of the accuracy of fungal protein synthesis by altering the conformation and activity of a ribosomal accuracy center, which is equivalent to the S4-S5-S12 ribosomal protein accuracy center domain of theE. coli ribosome. Furthermore, we suggest that EF-3 represents an evolving ribosomal protein with properties analogous to the intrinsic ATPase activities of higher eukaryotic ribosomes, which has wider implications for the evolutionary divergence of fungi from other eukaryotes. Correspondence to: M.F. Tuite     

Studies on the interaction between ribosomes and 14 CH 3 -F 3 initation factor

Summary Initiation factor F₃ has been purified fromEscherichia coli and labelledin vitro by reductive alkylation. The¹⁴CH₃–F₃ so obtained had a specific activity of about 1 000 cpm/g and was shown to have retained its biological activity. Labelled F₃ binds to 30S ribosomal subunits ofEscherichia coli andBacillus stearothermophilus, but does not bind to either 70S ribosomes or 50S ribosomal subunits. The stoichiometry of the binding indicates that one molecule of¹⁴CH₃–F₃ is bound to each 30S ribosomal subunit. Several antibiotics, known to interact with 30S subunits, inhibit the binding. Functional studies indicate that F₃ is released from 30S ribosomes as a result of the formation of the 70S initiation complex.     

Effective formaldehyde fixation of the reversibly interacting ribosomal system

The possiblity of using formaldehyde fixation for the quantitative studies of the reversibly interacting ribosomal system 50S–30S 50S+30S was investigated. Conditions have been found under which formaldehyde fixation of a reaction mixture of purified ribosomal particles proved to be possible without any change in the initial ratio of its components. This fixation technique was used in studying the dependence of the association level on Mg⁺⁺ ion concentration. An estimation has been made of the number of Mg⁺⁺ ions which bind during the association of 50S and 30S subparticles.     

Characteristics of ribonucleic acids isolated from Botryodiplodia theobromae pycnidiospores

Nucleic acids isolated from dormant and germinated Botryodiplodia theobromae pycnidiospores contain five distinct species of RNA. They include two ribosomal species, two ribosomal-associated species and transfer RNAs. Sedimentation coefficients of 25.1S and 18S were obtained for the two ribosomal RNA species and 5.8S and 5S for the two ribosomal-associated RNA components. Molecular weights of 1.20, 0.67, 0.054 and 0.035x10⁶ daltons were obtained after formaldehyde treatment and electrophoresis on polyacrylamide gels for these same four RNAs. Methylated nucleotides were present in the transfer RNAs and large and small ribosomal RNAs; in contrast 5.8S and 5S RNAs contained few methylated nucleotides. In addition to the 5 distinct RNA species, polyadenylate-containing RNA was isolated from both dormant and germinated spores.Published with the approval of the Director as paper no. 5006, Journal Series, Nebraska Agricultural Experiment Station. The work was conducted under Nebraska Agricultural Experiment Station Project no. 21-17.     

Utilization of 35S-Thiosulphate and an appraisal of the role of ATP-sulphurylase in chemolithotrophic Thiobacillus ferrooxidans

Differentially labelled ³⁵S-thiosulphate was taken up by washed cells of Thiobacillus ferrooxidans which were previously grown on thiosulphate. The uptake was proportional to the biomass over the range 0.5–4.0 mg dry wt. of bacteria and showed typical saturation kinetics with an estimated K _m value of 0.5 mM for ³⁵S-thiosulphate. Dithionate and Group VI anions inhibited the uptake, which was under pH control and had a temperature optimum of 50°C. In the absence of thiosulphate, the cells bound ³⁵S-sulphate but the binding did not increase on prolonged incubation and the label could be removed completely by washing with dilute sulphuric acid. Increasing amounts of the label were incorporated from [outer-³⁵S]thiosulphate into cellular materials over a 60-min period, whereas little or no assimilation was observed from either the [inner-³⁵S]thiosulphate or ³⁵S-sulphate. The kinetic properties of the sulphate-activating enzyme ATP_sulphurylase enriched from bacteria grown with either thiosulphate or ferrous-iron were similar although this enzyme has an assimilatory function only when the bacterium is grown with ferrous-iron.Abbreviation APS adenosine-5-sulphatophosphate     

Proteins occurring at, or near, the subunit interface of E. coli ribosomes

Summary The identification of ribosomal proteins that occur at, or near, the subunit interface of the 30S and 50S subunits in the E. coli 70S ribosome was attempted by studying the effect of antibodies on the Mg⁺⁺ dependent dissociation-association equilibrium of 70S ribosomes. Dissociated ribosomes were mixed with monovalent fragments of IgG antibodies (Fab's) specific for each ribosomal protein and then reassociated into intact 70S particles. Various degrees of inhibition of this reassociation were observed for proteins S9, S11, S12, S14, S20, L1, L6, L14, L15, L19, L20, L23, L26 and L27. A small amount of aggregation of 50S subunits was caused by IgG's specific for the proteins S9, S11, S12, S14 and S20 and purified 50S subunits. It was inferred that the presence of small amounts of these proteins on 50S subunits was compatible with their presence at the subunit interface. Finally, the capacity of proteins S11 and S12 to bind to 23S RNA was demonstrated.Paper No. 84 on Ribosomal Proteins. Preceding paper is by Rahmsdorf et al., Molec. gen. Genet. 127, 259–271 (1973).     

The arrangement of chromosomes in nuclei of sperm from plethodontid salamanders

Plethodontid salamanders have n = 13 or 14 large metacentric or sub-metacentric chromosomes. Sperm nuclei from Plethodon cinereus measure 72×1 m. The nucleoprotein of spermatids is at first finely granular. In elongate spermatids it clumps into larger granules, which then fuse to form the compact nucleoprotein of the mature sperm. The nuclei of mature sperm are negatively birefringent with respect to their length. — ³H RNA complementary to high-density satellite DNA of centromeric heterochromatin in P. cinereus has been hybridized in-situ to spermatids and sperm, and its site of binding to these cells has been examined by autoradiography. Labelling of round spermatid nuclei is localized in a single patch. Elongate spermatid nuclei are labelled only over the rear quarter of the nucleus. Label over the nuclei of mature sperm is localized in a region extending 10–20 m forwards from the rear of the nucleus. — In P. cinereus the ribosomal genes are located near the centromere on the short arm of chromosome 7. ³H ribosomal RNA hybridizes to a single patch in round spermatid nuclei. Elongate spermatid nuclei show label over a short segment of the rear half of the nucleus. In spermatids nearing maturity the labelled region is never more than 20 m long. — These results indicate that in P. cinereus each chromosome is arranged in a U formation with its centromere at the base of the sperm nucleus, and its arms extended forwards along the length of the nucleus. — Among plethodontids, increase in C value and corresponding increase in chromosome size is accompanied by increase in the length rather than the width of the sperm nucleus. — ³H ribosomal RNA hybridizes to a short segment in spermatid and sperm nuclei from Xenopus and Triturus. In these animals, the position of the labelled segment varies from sperm to sperm.     

Identification of a ∼30S size non-ribosomal Saccharomyces cerevisiae RNA that is rapidly labeled on its 3′ end by ATP or UTP

Cell-free extracts prepared from S. cerevisiae cells were incubated in the presence of [-³²P]-labeled ATP, CTP, GTP or UTP. An RNA larger than ribosomal 25S RNA with an apparent size of approximately 30S was prominently labeled on its 3 end in the presence of ATP or UTP but not with CTP or GTP. This labeled RNA was not hybrid-selected by cloned yeast ribosomal DNA; in addition, this 30S RNA was not cleaved by RNase H in the presence of complementary deoxyribooligonucleotides to rRNA. These two lines of evidence show that this 30S RNA is not structurally related to ribosomal RNA gene repeat. The cell-free extracts prepared from yeast cells containing temperature-sensitive poly(A) polymerase adenylated this novel yeast RNA at restrictive temperature with efficiency similar to extracts prepared from wild-type yeast cells. These data show that the enzyme responsible for adenylation of this 30S RNA is distinct from mRNA poly(A) polymerase. While the human SRP RNA 3 adenylating enzyme in the HeLa cell extract adenylated human SRP or Alu RNAs, the yeast adenylating enzyme did not adenylate the human SRP or Alu RNAs in vitro; these data indicate species specificity for this adenylating enzyme.     

Characterization of the ribosomal ribonucleic acids of blue-green algae

Summary The ribosomal RNA components of 12 species of blue-green algae have been characterized. The 23S RNA of most species is labile and discrete cleavage products were detected by polyacrylamide gel electrophoresis. In contrast, the 23S and 16S RNA's of three species, Anacystis nidulans, Nostoc sp. and Oscillatoria tenuis were essentially undegraded (apart from a hidden break in some of the 23S RNA molecules) and these are the most suitable species for further study. The undegraded 23S and 16S RNA's have similar molecular weights (1.07×10⁶ and 0.53–0.54×10⁶ respectively) to the corresponding molecules from bacteria and eukaryote chloroplasts. The nucleotide base compositions of separated, intact, 23S and 16S RNA's from blue-green algae are also of the prokaryotic type. For instance, the (G+C) content of each RNA is approximately 52 moles % and the (G-C)+(A-U) values are high (16–24 moles %). Blue-green algae, like other organisms, contain a 5S ribosomal RNA. Its electrophoretic mobility in polyacrylamide gels and its behaviour on methylated-albumen-kieselguhr-columns relative to E. coli, plant cytoplasmic and plant chloroplast 5S RNA's, are described.     

Kinetics of Incorporation of Uridine-C14 into L Cell RNA

Five components have been isolated from L cells by a combination of phenol extraction procedures and sedimentation analysis through sucrose gradients. These components are identified by their sedimentation rates. The 50S and 40S components are derived from the nucleus, the 32S and 18S from ribosomal RNA, and the 4S fraction is the soluble RNA of the cell. L cells were supplied with uridine-C¹⁴ under steady-state conditions and the rate of uptake of C¹⁴ into each component was measured. Analysis of the results suggests that the delay in entry of C¹⁴ into ribosomal RNA is occasioned by two sequential precursors and that 50S and 40S RNA meet the kinetic requirements for these precursors. 4S RNA seems to contain two components that label at different rates.     

Differential localization of ribosomal proteins S14 and 7/8 in egg chambers of D. melanogaster

Summary The localization of Drosophila melanogaster ribosomal proteins S14, and 7/8 during oogenesis was studied by indirect immune fluorescence microscopy. The acidic proteins S14¹ and 7/8¹ were isolated from D. melanogaster embryonic ribosomal proteins by carboxymethylcellulose chromatography (Chooi 1980). Antibodies raised against each of these proteins were applied to ovariole squashes, and the position of each antibody was localized by fluorescein labeled sheep antirabbit IgGs. Anti-S14 was found predominantly in nurse cell nuclei, follicle cell nuclei, oocytes and, to a much lesser degree, in nurse cell and follicle cell cytoplasm. In contrast, anti-7/8 was found in major quantities in nurse cell and follicle cell cytoplasm, and oocytes. Anti-7/8 in the nurse cell and follicle cell nuclei was either not detectable or at a strikingly lower level than that found in the corresponding cytoplasm. The egg chamber patterns of localization of these two proteins were also found in salivary gland cells. However, in Drosophila tissue culture cells, these patterns were altered; both anti-S14 and anti-7/8 were detected only in the cytoplasm.     

Heterogeneity of ribosomal populations in Escherichia coli cells grown in different media

Summary In order to establish whether ribosomes exhibit heterogeneity with respect to their protein pattern in vivo, E. coli cells were grown in rich or minimal medium and labeled with ¹⁴C and ³H amino acid mixture, respectively. After harvesting, the cells from the different media were mixed, the differently labeled ribosomes isolated and the ribosomal proteins separated. For each protein the ratio of ¹⁴C to ³H was determined and used as an indication of whether differences exist in ribosomal populations synthesized under different growth conditions.With respect to their ratio the ribosomal proteins can be classified as follows: Many of the proteins have a ratio of 1, i. e. they are present in the same amount in both preparations. The ratios for about 30% of the proteins differ only slightly from 1 whereas three proteins namely S6, S21 and L12 have ratios of 2.5 and 3.1 respectively. This means that ribosomal populations isolated from cells grown in rich medium contain these three proteins in two to three fold greater amounts compared to those synthesized in minimal medium.The relevance of these results with respect to the occurrence of heterogeneous ribosomal populations in vivo is discussed.Paper Nr. 36 on Ribosomal Proteins. Preceding paper is by H. J. Weber, Mol. Gen. Genetics 119, 233–248 (1972).     

Particle-bound phytochrome: Association with a ribonucleoprotein fraction from Cucurbita pepo L.

Summary In the absence of ethylenediaminetetraacetic acid (EDTA) and added Mg²⁺, the phytochrome, RNA, protein, cytochrome c oxidase and NADPH-cytochrome c reductase in 20000 x g pellets from hypocotyl hooks of red-irradiated Cucurbita seedlings are more or less coincident in a single, broad band on linear sucrose gradients. The inclusion of 3 mM EDTA in the extraction, resuspension and gradient media has three major effects: (a) The phytochrome profile splits into two main bands; (b) the main RNA population shifts to a sharp peak which co-sediments with the lighter phytochrome band at 31S; (c) the main NADPH-cytochrome c reductase peak shifts to a lower density. This indicates that the EDTA dissociates a rough-endoplasmic-reticulum fraction into separate membrane and ribonucleoprotein (RNP) components, and that part of the phytochrome is associated with the latter. The 31S RNP fraction is 35–40% RNA, has a 260/235 nm absorption ratio of 1.36 and the RNA dissociates into small fragments in sodium dodecyl sulfate. More than 90% of the phytochrome and RNA in the isolated 31S fraction becomes pelletable upon the addition of 10 mM Mg²⁺. Higher Mg²⁺ levels release the phytochrome and some of the other protein present from the RNA which remains pelletable. The data indicate that the 31S RNP fraction may be degraded ribosomal material with extraneously bound protein, including phytochrome. Several aspects of phytochrome-binding to particulate fractions which have been reported in the literature are consistent with an interaction of P_fr with ribosomal material—degraded or otherwise.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - RNase ribonuclease - RNP ribonucleoprotein - SDS sodium dodecyl sulfate