Evidence for differential regulation of two classes of poly(A) RNA inBlastocladiella emersonii zoospores

The poly(A) RNA in zoospores ofBlastocladiella emersonii contains RNA synthesized during the growth phase (GP poly(A) RNA) and late sporulation (LS poly(A) RNA). LS poly(A) RNA synthesized during the final 30 minutes of sporulation is bound exclusively to polyribosomes which comprise approximately 50% of the total zoospore ribosome population. In contrast, GP poly(A) RNA is bound to zoospore monoribosomes. During the final 30 minutes of sporulation, GP poly(A) RNA which is bound to polyribosomes makes a transition to monoribosomes. Zoospore monoribosomes and RNA extracted from zoospore monoribosomes are inactivein vitro while both zoospore polyribosomes and RNA extracted from zoospore polyribosomes stimulate protein synthesis in the wheat germin vitro system. Sedimentation of poly(A) RNA from zoospore monoribosomes on dimethyl sulfoxide gradients revealed that the GP poly(A) RNA was of sufficiently high molecular weight to code for average-sized proteins. These denaturing gradients failed to activate the zoospore monoribosome RNA. The results suggest that the inability to translate zoospore monoribosomesin vitro is due to some property or modification of the zoospore monoribosome poly(A) RNA. Zoospore monoribosomes bound to poly(A) RNA contain an average of two tRNA molecules while zoospore polyribosomes have an average of less than one tRNA bound. This suggests the two classes of ribosomes are blocked at different steps in the elongation process.     

Structure of methemerythrin at 5 A resolution.

Rabbit globin messenger RNA was labelled in vitro with ¹²⁵I to specific activities in the range 20 to 200 × 10⁶ cts/min per μg. This ¹²⁵I-labelled mRNA bound to rabbit reticulocyte ribosomes with the kinetics and sensitivity to inhibitors expected from its participation in the normal process of the initiation of protein synthesis. Furthermore, when modified in 25% of its cytidine residues with unlabelled iodide, the mRNA coded for the same series of initiation peptides as did the unmodified mRNA. Using the techniques of RNA fingerprinting, the binding reaction was shown to select against contaminants and against “globin mRNA” molecules which lack a particular oligonucleotide implicated in the initiation process. When the ¹²⁵I-labelled mRNA was bound to ribosomes, both the initiating 40 S subunits and the 80 S ribosomes protected a fraction of the mRNA from digestion by pancreatic ribonuclease. Fingerprint analysis showed that highly specific regions of the mRNA were protected by the 40 S subunits and 80 S ribosomes and that these two protected regions were not identical.     

The isolation and properties of cardiac ribosomes and polysomes

1. A method is described by which good yields of ribosomes and polysomes free of contamination by submitochondrial fragments can be prepared from rat cardiac muscle. These preparations are capable of incorporation of amino acids into protein in vitro. 2. The ribosome preparation consists of 32% of monomeric ribosomes and 68% of ribosomal aggregates or polysomes. The polysome preparation has a decreased monomeric content. Dimers, trimers, tetramers, pentamers and larger components can be differentiated. 3. The polysome aggregate structure is degraded to monomeric ribosomes on incubation with small amounts of ribonuclease or by preparation in the absence of Mg²⁺ ions. The degradation in the absence of Mg²⁺ ions was not reversible and drastically decreased the incorporation of amino acids in vitro. 4. The cardiac ribosomes contained two major RNA species sedimenting at 19s and 28s in a 1:2·4 ratio. 5. The RNA/protein ratio of cardiac ribosomes and polysomes was consistently lower than that of similar preparations from liver. The concentrations of Na⁺ and K⁺ ions present during preparation had a great effect on the RNA/protein ratio. 6. Optimum conditions for the incorporation of amino acids into protein in vitro are reported. Cardiac ribosomes have a lower rate of incorporation of amino acids in vitro than liver ribosomes. 7. Heart cell sap is less active than liver cell sap: evidence is presented that a factor, present in liver cell sap and concerned with stimulating the synthesis of the peptide chain, is lacking in heart cell sap. 8. Pulse-labelling of perfused hearts followed by examination of the subcellular structures showed that the ribosomal fraction was the most active in the incorporation of amino acids in vitro.     

Protein synthesis in Bacillus subtilis. I. Hydrodynamics and in vitro functional properties of ribosomes from B. subtilis W168.

On the basis of their sedimentation properties, the ribosomal particles in crude extracts of Bacillus subtilis W168 are characterized as pressure-sensitive couples, pressure-resistant couples, or non-associating subunits. Pressure-sensitive couples dissociate into subunits, yielding a peak at 60 S in the gradient profile, on sedimentation at high speed in the presence of 10 to 15 mm-Mg²⁺. Under the same conditions, pressure-resistant couples sediment at 70 S. Under certain conditions, pressure-resistant couples apparently aggregate, possibly in 70 S · 70 S dimers. Procedures are described for the isolation of pressure-sensitive couples from B. subtilis. The isolated couples are shown by chemical fixation experiments to require approximately twice the Mg²⁺ concentration required by Escherichia coli couples to remain associated at atmospheric pressure.All three types of B. subtilis ribosome incorporate amino acids into acid-insoluble material in the presence of B. subtilis cellular RNA, B. subtilis ribosomal salt wash fraction, and E. coli post-ribosomal supernatant. Overall incorporation, dependence on added RNA, and dependence on salt wash fraction are greatest with pressure-sensitive couples. The products of protein synthesis in vitro stimulated by total B. subtilis RNA appear to be a low molecular weight subset of the proteins synthesized most abundantly in vivo. Incubation of pressure-sensitive couples with cellular RNA from B. subtilis, fMet-tRNA_f^Met, ribosomal salt wash fraction and GTP results in their conversion to pressure-resistant couples, with concomitant and stoichiometric binding of fMet-tRNA to the 70 S species. It is concluded that in B. subtilis as in E. coli, pressure-sensitive couples are “vacant”, while pressure-resistant couples are “complexed” with messenger RNA. fMet-tRNA-bearing complexed couples are interpreted as initiation complexes in which ribosomes have bound mRNA, presumably at initiation sites. Their formation in vitro is strictly dependent on RNA, salt wash fraction and fMet-tRNA when vacant ribosomal couples are used.     

An electron microscopic analysis of pathways for bacteriophage T4 DNA recombination

The interaction between ribosomes of Bacillus stearothermophilus and the RNA genomes of R17 and Qβ bacteriophage has been studied. Whereas Escherichia coli ribosomes can initiate the synthesis of all three RNA phage-specific proteins in vitro, ribosomes of B. stearothermophilus were previously shown to recognize only the A (or maturation) protein initiation site of f2 or R17 RNA. Under these same conditions, a Qβ region is bound and protected from nuclease digestion. Qβ RNA, however, does not direct the synthesis of any formylmethionyl dipeptide in the presence of B. stearothermophilus ribosomes, nor does the binding of either this Qβ region or the R17 A protein initiation site to these ribosomes show the same fMet-tRNA requirement for recognition of initiator regions as that previously established with E. coli ribosomes. Analysis of a 38-nucleotide sequence in the protected Qβ region reveals no AUG or GUG initiator codon. These observations suggest that messenger RNA may be recognized and bound by B. stearothermophilus ribosomes quite independently of polypeptide chain initiation.Binding experiments using R17 RNA and mixtures of components from B. stearothermophilus and E. coli ribosomes confirm the conclusion drawn by Lodish (1970a) that specificity in the selection of authentic phage initiator regions by the two species resides in the ribosomal subunit(s). However, anomalous attachment of B. stearothermophilus ribosomes to R17 RNA, which is observed upon lowering the incubation temperature of the binding reaction, is clearly a property of the initiation factor fraction. The results are discussed with respect to current ideas on the role of ribosomes and initiation factors in determining the specificity of polypeptide chain initiation.     

Protein synthesis in Bacillus subtilis. II. Selective translation of natural mRNAs and its possible relation to the species-specific inhibition of protein synthesis by lincomycin and erythromycin.

Vacant ribosomal couples from Bacillus subtilis W168 incorporate only very small amounts of amino acids into polypeptides in response to Escherichia coli cellular RNA or bacteriophage f2 RNA, but are observed to form initiation complexes in the presence of f2 RNA. Vacant ribosomal couples from E. coli acquire pressure-resistance, but do not bind fMet-tRNA, when incubated with B. subtilis RNA in the absence of ribosomal wash fraction. The implied mRNA binding in the absence of salt wash fraction, taken with previously reported observations of salt wash-independent translation of mRNAs from Grampositive bacteria, suggests that mRNAs from Gram-positive bacteria have an active functional character which is masked or absent in mRNAs from Gram-negative sources. It is proposed that this property of B. subtilis mRNAs is required by B. subtilis ribosomes for some translational function subsequent to the formation of the 70 S initiation complex, and that f2 RNA, while it is bound by B. subtilis ribosomes in initiation complexes, is not translated because it lacks this feature.The antibiotic lincomycin has been found to inhibit translation of natural mRNAs in vitro in systems from Gram-positive bacteria at concentrations 10 to 100 times lower than those necessary to inhibit translation in systems from Gram-negative species. Lincomycin does not inhibit formation of initiation complexes by vacant couples from B. subtilis or E. coli. Taken with the published findings of other investigators, these results are interpreted as indicating that the first translocation step following assembly of the initiation complex may coincide with a transition between distinct “initiating” and “elongating” states of the ribosome, and that this transition may involve structural elements, and possibly mechanisms, which are different in Gram-positive systems than in Gram-negative systems.A comprehensive model is constructed to account for the results of these studies and for the published findings of other investigators. It is proposed that some feature of Gram-positive mRNA, perhaps a vestige of early protein synthetic systems, is required by the ribosomes of Gram-positive bacteria to facilitate the transition between initiating and elongating ribosomal states. Inhibition of protein synthesis by lincomycin and the similarly species-specific macrolide antibiotic erythromycin is interpreted as an allosteric effect on the transition between initiating and elongating ribosomal states, in which the different binding affinities of ribosomes from Gram-positive and Gram-negative bacteria for the drugs are related to the functional differences between the two types of systems at this critical step. The implications of this interpretation of interspecies translational specificity for mechanisms of translational control in the cell and for the nature of the divergence of bacterial protein synthesis systems into Gram-positive and Gram-negative types are discussed.     

Decreased activity of Blastocladiella emersonii zoospore ribosomes: correlation with developmental changes in ribosome-associated proteins

Ribosomal proteins isolated from dormant zoospores were compared to the ribosomal proteins found in the active growth phase by two-dimensional polyacrylamide gel electrophoresis. Zoospore ribosomes were found to contain a set of five proteins, designated Z1 to Z5, which were not present in growth phase ribosomes. The Z1-Z5 proteins were not removed by high-salt washes using either 1 M KCl or 1 M NH4 Cl. The Z1 protein is found associated with zoospore 60 S subunits while Z2-Z5 are bound to 40 S subunits. Zoospore monoribosomes and polyribosomes contain comparable levels of each of the five proteins. Approximately 60 min. after sporulation is induced, the Z1-Z5 proteins begin to accumulate on the ribosomes with the highest levels of these proteins found associated with ribosomes at the zoospore stage. During germination, the proteins gradually disappear and are not detectable on the ribosomes after 4 hr of germination. The presence of the Z1-Z5 proteins correlates with a decrease in in vitro protein synthetic activity of the fungal ribosomes. The data are consistent with the hypothesis that the proteins regulate translation by completely blocking protein synthesis on a subset of ribosomes while the remainder of the ribosomes function at normal rates.     

The Influence of Axis Removal on Protein Metabolism in Cotyledons of Pisum sativum L

The protein metabolism of cotyledons attached to the embryonic axis has been compared with that in cotyledons removed from the axis at the initiation of a 6-day imbibition. Total protein declined in the attached but not in the detached cotyledons. Concurrent with the decline in protein level in the intact cotyledons there was an increased capacity to incorporate exogenously supplied leucine into protein. In contrast, detached cotyledons showed a restricted capacity for protein synthesis. It was demonstrated that ribosomal preparations from cotyledons of intact seedlings contained an increasing proportion of polyribosomes as germination progressed and such ribosomes were active in in vitro amino acid incorporation. Ribosomal preparations from detached cotyledons contained few polyribosomes and had a restricted capacity to incorporate amino acids in vitro. The in vitro incorporation of phenylalanine was stimulated by polyuridylic acid with the stimulation being greatest in ribosomal preparations from detached cotyledons. The results suggest that an axis component may regulate the availability of messenger RNA in the cotyledons during germination.     

Specificity of protein synthesis by bacterial ribosomes and initiation factors: Absence of change after phage T4 infection

Using several natural messenger RNA's—f2 RNA, Qβ RNA, T7 RNA, T4 early mRNA, T4 late mRNA and Escherichia coli RNA—ribosomes isolated from cells either 5 or 12 minutes after T4 infection direct synthesis of only 35 to 70% as much protein as do ribosomes from uninfected cells. However, with poly(U) or formaldehyde-treated f2 RNA message, both types of ribosomes work equally well. Experiments mixing salt-washed ribosomes and initiation factors from these cells show, in agreement with work of others, that the reduction with natural messages is due only to changes in the initiation factors.     

Regulation of Protein Synthesis in Zoospores of Blastocladiella

The factors responsible for the regulation of protein synthesis in the zoospores of Blastocladiella emersonii were studied by means of cell fractionation and in vitro assays. Charged transfer ribonucleic acid (tRNA) and aminoacyl-tRNA synthetases were found both inside the membrane-bound, ribosomal nuclear cap, and in the extracap cytoplasm. Ribosomes isolated from zoospore nuclear caps in low salt buffer failed to support polyuridylic acid-dependent phenylalanine incorporation. After washing with high salt buffer, the cap ribosomes were equivalent in activity to similarly prepared plant ribosomes. Both the high-salt wash from cap ribosomes and the extracap supernatant fraction contained an unidentified material which inhibited aminoacyl-tRNA binding and peptide bond formation by ribosomes. Ribosomal binding of polyuridylic acid was not inhibited. Washed cap ribosomes supported very low incorporation rates without added messenger RNA, and were highly dependent upon added poly U for phenylalanine incorporation, indicating a low level of messenger in nuclear caps. It is concluded that enclosure of the ribosomes in the nuclear cap does not in itself prevent protein synthesis, and that the lack of activity may be due to the presence of a ribosome inhibitor.     

Structural dynamics of bacterial ribosomes. I. Characterization of vacant couples and their relation to complexed ribosomes

Ribosomes not engaged in protein synthesis (vacant couples), in contrast to complexed ribosomes bearing nascent chains, dissociate during sedimentation in sucrose gradients at high g forces and at Mg²⁺ concentrations below 15 mm. As a result of this dissociation, a new peak between the 70 S complexed ribosomes and the free 50 S subunits is observed, the position of which shifts from about 55 S to 70 S as the Mg²⁺ concentration in the gradient is raised from 5 to 15 mm. The apparent 60 S peak consists of 50 S subunits produced during dissociation in the gradient. At low g forces, the sedimentation rate of complexed and vacant ribosomes is indistinguishable, even at 5 mm-Mg²⁺. These sedimentation properties are valid criteria to differentiate vacant and complexed ribosomes. This is shown by converting complexed ribosomes quantitatively into vacant couples by removing the nascent chains through termination release or with puromycin, or by converting vacant couples into initiation complexes with R17 RNA, fMet-tRNA and initiation factors.Ribosomes from cells harvested by slow cooling consist almost entirely of vacant couples, all of which are active in protein synthesis with natural messengers. The structural features responsible for the interaction between subunits are discussed.     

Regulation of protein synthesis in vesicular stomatitis virus-infected mouse L-929 cells by decreased protein synthesis initiation factor 2 activity.

Infection of mouse L-cell spinner cultures by vesicular stomatitis virus (VSV) effected the selective translation of viral mRNA by 4h after viral adsorption. Cell-free systems prepared from mock- and VSV-infected cells reflected this phenomenon; protein synthesis was reduced in the virus-infected cell lysate by approximately 75% compared with the mock-infected (control) lysate. This effect appeared to be specific to protein synthesis initiation since (i) methionine incorporation into protein from an exogenous preparation of initiator methionyl-tRNA gave completely analogous results and (ii) the addition of a ribosomal salt wash (containing protein synthesis initiation factors) stimulated protein synthesis by the infected cell lysate but had no effect on protein synthesis by the control. Micrococcal nuclease-treated (initiation-dependent) VSV-infected cell lysates were not able to translate L-cell mRNA unless they were supplemented with a ribosomal salt wash; a salt wash from ribosomes from uninfected cells effected a quicker recovery than a salt wash from ribosomes from infected cells. When salt wash preparations from ribosomes from uninfected and infected cells were tested for initiation factor 2 (eIF-2)-dependent ternary complex capacity with added GTP and initiator methionyl-tRNA, we found that the two preparations contained equivalent levels of eIF-2. However, initiation complex formation by the factor from virus-infected cells proceeded at a reduced initial rate compared with the control. When the lysates were supplemented with a partially purified eIF-2 preparation, recovery of activity by the infected cell lysate was observed. Mechanisms by which downward regulation of eIF-2 activity might direct the selective translation of viral mRNA in VSV-infected cells are proposed.     

Phage-Displayed Peptides as Developmental Agonists for Phytophthora capsici Zoospores

As part of its pathogenic life cycle, Phytophthora capsici disperses to plants through a motile zoospore stage. Molecules on the zoospore surface are involved in reception of environmental signals that direct preinfection behavior. We developed a phage display protocol to identify peptides that bind to the surface molecules of P. capsici zoospores in vitro. The selected phage-displayed peptides contained an abundance of polar amino acids and proline but were otherwise not conserved. About half of the selected phage that were tested concomitantly induced zoospore encystment in the absence of other signaling agents. A display phage was shown to bind to the zoospore but not to the cyst form of P. capsici. Two free peptides corresponding to active phage were similarly able to induce encystment of zoospores, indicating that their ability to serve as signaling ligands did not depend on their exact molecular context. Isolation and subsequent expression of peptides that act on pathogens could allow the identification of receptor molecules on the zoospore surface, in addition to forming the basis for a novel plant disease resistance strategy.     

Ribonucleic acid and protein synthesis in Rhizophlyctis rosea zoospores

LéJohn, Herbert B. (Purdue University, Lafayette, Ind.), and James S. Lovett. Ribonucleic acid and protein synthesis in Rhizophlyctis rosea zoospores. J. Bacteriol. 91:709-717. 1966.-The uniflagellate zoospores of Rhizophlyctis rosea display active motility and a high endogenous respiratory metabolism, but neither growth nor net ribonucleic acid (RNA) or protein synthesis can be measured by ordinary procedures. Nevertheless, synthesis can be detected with isotopic precursors. Uracil-C(14) is incorporated slowly into both the soluble and ribosomal RNA. Analysis of zoospore extracts (on diethylaminoethyl cellulose columns or sucrose gradients) after various periods of labeling suggested that most of the uracil incorporation represents slow synthesis of ribosomal precursor RNA and, ultimately, ribosomes. Actinomycin D caused an 80% inhibition of uracil incorporation. The most rapidly labeled RNA was susceptible to extensive degradation in cells treated with actinomycin, but the percentage of stable RNA increased with the time of incorporation before addition of the antibiotic. Neither the effects of actinomycin nor the results of chase experiments have established unequivocally the existence of turnover or the presence of a short-lived "messenger" fraction in motile spores. Both leucine and methionine were slowly incorporated into a spectrum of cellular proteins. The methyl group of C(14)-methylmethionine also served as a methyl donor for the methylation of soluble RNA but not of ribosomal RNA. The observations that some of the newly synthesized RNA and protein occur in the intact 82S ribosomes and that actinomycin inhibits the low level of protein synthesis provide some indirect evidence for a very low rate of "messenger" synthesis and turnover in zoospores.     

Recessive nonsense-suppression in yeast Saccharomyces cerevisiae

Summary The shift of recessive suppressor mutant of yeast Saccharomyces cerevisiae from permissive to restrictive conditions is accompanied by polysome decay and accumulation of 80 S ribosomes (Smirnov et al., 1976). In this paper some properties of 80 S ribosomes are studied. It is demonstrated that polysome decay under non-permissive conditions is not the consequence of the impairement of RNA synthesis. More than 70% of 80 S ribosomes accumulated under non-permissive conditions contain bound peptidyl-tRNAs localized in P-ribosomal site. tRNA moiety of bound peptidyl-tRNA is able to accept all 20 natural amino acids after chemical deacylation. Therefore it is not a specific isoacceptor species but rather total tRNA that is bound to ribosomes. The polypeptide residues of these peptidyl-tRNAs are heterogeneous in size. Their molecular weights are comparable with the molecular weights of the completed polypeptides. Some of the 80 S ribosomes accumulated under non-permissive conditions contain poly-A RNA. In conclusion, possible mechanism of the impairement of translation under non-permissive conditions in recessive suppressor strain is discussed.     

Polyamines are necessary for maximum in vitro synthesis of globin peptides and play a role in chain initiation.

The salt wash fraction removed from rabbit reticulocyte ribosomes with 0.5 m KCl contains dialyzable components required for maximum in vitro synthesis of globin peptides. The active substances were identified as spermidine and spermine. Rabbit reticulocyte ribosomes contain spermine and spermidine in a 1:3 ratio of which about 75% is removed in the 0.5 m KCl wash fraction. Dialyzed salt wash can be reactivated for in vitro protein synthesis by addition of either spermine, spermidine, or Mg²⁺ ion. A twofold higher leucine incorporation into protein was obtained with the optimum concentration of either polyamine than with Mg²⁺. Spermidine is effective in lowering the Mg²⁺ requirement for initiation of phenylalanine peptides in the poly(U)-directed system, apparently by formation of an initiation complex. Also, spermidine competitively interferes with edeine inhibition of globin chain initiation. These results indicate that spermidine may play a special role in peptide initiation.     

Use of antibiotics to determine ribosome-messenger RNA interactions necessary for in vivo stability of specific messengers

With the use of inhibitors of individual reactions in protein synthesis, a method has been developed for (a) determining the role of ribosome messenger RNA interactions in specific bacteriophage T4 mRNA stability and (b) localizing the primary site of interaction of messenger ribonuclease (mRNase) on messengers. Antibiotics that freeze ribosomes in or near the initiation site stabilize T4 deoxynucleotide kinase mRNA. In contrast, T4 α-glucosyltransferase mRNA is stable only when the polysome configuration is kept intact. These results indicate the deoxynucleotide kinase mRNA initiation site is most susceptible to mRNase action, whereas the primary site of mRNase action on the α-glucosyltransferase mRNA is distal to the initiation site. Determining the role of ribosome-mRNA interactions in mRNA stability by the use of specific inhibitors of protein synthesis may be applicable to any procaryotic or eucaryotic mRNA that can be translated in vitro.     

Regulation of protein synthesis in mitotic HeLa cells.

Mitotic HeLa cells (M cells) synthesize protein at about 25% of the rate of S phase cells. This decrease in protein synthesis is due to a reduction in the rate of initiation. However, extracts prepared from M cells are almost as active in protein synthesis as S cell extracts. Both cell extracts are quite active in in vitro initiation of protein synthesis. Moreover, two steps in initiation, binding of Met-tRNAf to 40S ribosomal subunits and binding of mRNA to ribosomes, show similar activity in both extracts. The difference in protein synthesizing activity observed in vivo is largely eliminated in the preparation of cell-free systems. The ribosomes of M cells contain small mol wt RNA, which inhibits protein synthesis in vitro. This RNA, which has possibly a nuclear origin, may be a cause of the reduction in the rate of protein synthesis in M cells.     

Inhibitors of cytoplasmic protein synthesis purified from rat liver mitochondria

Summary Purified mitochondria from rat liver were found to contain protein synthesis inhibitors, that could be extracted by disruption of mitochondrial membranes and fractionated by gel filtration into two fractions of low and high molecular weight. Small size inhibitors were also released from the latter peak by high ionic strength followed by gel filtration. Both types of factors inhibit incorporation of radioactive amino acids into protein by liver cytoplasmic polysomes programmed with endogenous mRNA or poly U, and by rabbit reticulocyte lysates programmed with added globin mRNA and by incubations of Walker carcinoma cells. They decrease to the same level the cytoplasmic synthesis of proteins for the mitochondrial and extra-mitochondrial compartments in intact cells, but do not appear to inhibit substantially endogenous mitochondrial protein synthesis. Inhibitors were purified by paper chromatography and reverse phase high performance liquid chromatography into fractions which block with the same kinetics the incorporation of [¹⁴]leucine and [³⁵]methionine into protein in systems able to initiate protein synthesis, such as reticulocyte lysates or intact cells, but differ in this respect in incubations of liver ribosomes where re-binding of mRNA is a limiting step. Some of these factors behave as oligopeptides that are assumed to inhibit in vitro primarily the initiation stage but whose function in vivo is still undetermined.