THE RELATIONSHIP BETWEEN RNA SYNTHESIS AND HEMOGLOBIN SYNTHESIS IN AMPHIBIAN ERYTHROPOIESIS : Cytochemical Evidence

A cytochemical study of the relationship between RNA synthesis and hemoglobin synthesis has been performed on splenectomized newts, Triturus viridescens. Employing radioautography, labeled cytidine was incorporated into the RNA of the early developmental stages but was not incorporated in the later stages. Labeled leucine was incorporated into the cellular protein of all stages except mature erythrocytes but was incorporated at a higher level in the later stages. Microphotometric measurements of azure B binding to cytoplasmic RNA revealed a sharp initial increase between the stem cell and proerythroblast followed by a rapid decrease between the basophilic and polychromatophilic stages. The loss of cytoplasmic RNA became more gradual in the late stages and, in the mature erythrocyte, little or no cytoplasmic RNA could be detected. Measurements of cytoplasmic total protein, using fast green staining at pH 2.0, and of heme showed that both curves increased similarly with development, indicating net hemoglobin synthesis. The results are compatible with the hypothesis that, as the stem cell differentiates along erythrocytic lines, a stable "messenger" RNA specifying the production of a given type or types of hemoglobin is formed. This complex probably becomes associated with ribosomal RNA and is retained throughout the process of RBC differentiation.     

Stage-specific initiation factors for protein synthesis during insect development

In insects, as in bacteria, the smaller (40 S) ribosomal subunit binds messenger RNA during initiation of protein synthesis. An 80 S ribosomal unit is formed by association of free 40 S and 60 S subunits. Formation of the complete initiation complex requires GTP, aminoacyl-tRNA, protein initiation factors and messenger RNA. The complex sediments as an 80 S band on sucrose gradient. Protein initiation factors are extracted from unwashed ribosomes and appear to be able to discriminate between messenger RNAs obtained from different stages of development. They promote formation of the 80 S complex only when messenger RNA is extracted from the same stage of development, providing a mechanism for control of protein synthesis by which ribosomes can select the messenger RNA to be translated. Two possibilities have been proposed to explain this phenomenon: (1) that a group of messenger RNAs from a given stage of development may have a specific sequence of nucleotides preceding the AUG codon. This sequence is recognized by a stage-specific element of the initiation machinery; (2) and or, the secondary structure of messenger RNA from a given stage of development may be specific and therefore recognized by a unique initiation factor.     

Synchronized development of gametophytic germlings of the aquatic PhycomyceteAllomyces macrogynus

Summary Gametophytic germlings ofAllomyces macrogynus are examined with regard to the sequence of developmental events which have been suggested to be effected by long-lived messenger RNA. During encystment of the meiospore, the cytoplasmic triplet microtubules are depolymerized, the gamma bodies are mobilized, the nuclear cap envelope is fragmented and the cap ribosomes are dispersed, the side body complex breaks into its components, and a cyst wall is deposited. All these processes take place prior to the appearance of significant amounts of protein synthesis. The first fission of the mitochondria occurs shortly after the onset of protein synthesis but before detectable RNA synthesis. The developmental sequence of gametophytic and sporophytic germlings was found to be very similar.     

Endogenous messenger ribonucleic acid-directed polypeptide chain elongation in a cell-free system from the yeast Saccharomyces cerevisiae.

An in vitro protein-synthesizing system from the yeast Saccharomyces cerevisiae has been made by a modification of the procedure for preparation of the Krebs ascites system. The protein synthetic activity is directed by endogenous messenger. Amino acid incorporation occurs over a broad range of magnesium and potassium concentration, being maximal at 6 and 85 mM, respcetively. The activity of this in vitro system is due to the elongation of polypeptides whose synthesis was initiated in vivo. The cell extract does not initiate synthesis with endogenous messenger ribonucleic acid (RNA), since 1 muM pactamycin, which blocks initiation on prokaryotic or eukaryotic ribosomes invitro, fails to decrease amino acid incorporation. Ten micromolar cycloheximide, however, inhibits incorporation by 87%. Moreover, this system is not stimulated by rabbit reticulocyte polysomal RNA, which directs the synthesis of hemoglobin in extracts of Krebs ascites cells. The translation of this messenger is not masked by high endogenous incorporation, because autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing [35-S]methionine-labeled products shows that no hemoglobin is made. Preincubation of this system, which reduces the high endogenous incorporation by 80%, does not increase its capacity to be stimulated by either rabbit reticulocyte RNA or yeast polyriboadenylic acid-containing RNA. Polyuridylic acid, however, does stimulate polyphenylalanine incorporation. The failure of the yeast lysate to be stimulated by or to translate added natural messenger RNA, its insensitivity to low levels of pactamycin but inhibition by cycloheximide, and its relatively high magnesium optimum (the same as that for polyuridylic acid) suggest that it elongates but does not initiate polypeptide chains.     

Streptomycin Action: Greater Inhibition of Escherichia coli Ribosome Function with Exogenous than with Endogenous Messenger Ribonucleic Acid

Inhibition of protein synthesis by streptomycin was tested in extracts from a strain of Escherichia coli sensitive to streptomycin. Three kinds of messenger ribonucleic acid (RNA) were employed: endogenous cellular RNA, extracted cellular RNA, and phage R17 RNA. Protein synthesis directed by extracted cellular RNA was inhibited three- to fourfold more than protein synthesis directed by endogenous RNA. With R17 RNA as messenger, nearly total inhibition of protein synthesis at initiation was again observed. The greater inhibition of function of extracted RNA, which must initiate new polypeptide chains in vitro, is in accord with the observation that in whole cells streptomycin blocks ribosomes at an early stage in protein synthesis. When streptomycin was added at successively later times during protein synthesis, the subsequent inhibition was progressively less. This was observed with either extracted cellular RNA or phage R17 RNA. A model is presented that can explain the less drastic inhibition by streptomycin of messenger RNA that is already functioning on ribosomes.     

Rates of synthesis and degradation of ribosomal ribonucleic acid during differentiation of Dictyostelium discoideum.

Synthesis of ribosomes and ribosomal ribonucleic acid (RNA) continued during differentiation of Dictyostelium discoideum concurrently with extensive turnover of ribosomes synthesized during both growth and developmental stages. We show here that the rate of synthesis of 26S and 17S ribosomal RNA during differentiation was less than 15% of that in growing cells, and by the time of sorocarp formation only about 25% of the cellular ribosomes had been synthesized during differentiation. Ribosomes synthesized during growth and differentiation were utilized in messenger RNA translation to the same extent; about 50% of each class were on polyribosomes. Ribosome degradation is apparently an all-or-nothing process, since virtually all 80S monosomes present in developing cells could be incorporated into polysomes when growth conditions were restored. By several criteria, ribosomes synthesized during growth and differentiation were functionally indistinguishable. Our data, together with previously published information on changes in the messenger RNA population during differentiation, indicate that synthesis of new ribosomes is not necessary for translation of developmentally regulated messenger RNA. We also establish that the overall rate of messenger RNA synthesis during differentiation is less than 15% of that in growing cells.     

Macromolecular Changes Associated with the Growth of Crustacean Tissues

Tissue mass, rate of protein synthesis, content of ribosomalRNA and rates of synthesis of ribosomal RNA have been studiedthroughout the molting cycle in the midgut gland, epithelium,and somatic muscle in the land crab, Gecarcinns lateralis. Inall tissues there is an increase in ribosomal RNA followed byan increase in the rate of synthesis of protein in the premoltperiod. Subsequently, the three tissues differed in that (a)in the midgut gland the level of ribosomal RNA and protein synthesisreturned to the intermolt rates before ecdysis whether or notthe mass of the tissue was increasing or decreasing; (b) ribosomalRNA and protein synthesis in epithelium reached a maximum ata time when epithelial cells reached a maximal size; subsequently,all three parameters decreased toward intermolt levels beforeecdysis; (c) ribosomal RNA and protein synthesis reached a maximumin the premolt period in somatic muscle while the muscle wasin fact decreasing in mass. Muscle ribosomes are very stableand appear to be conserved for weeks or months to be reusedafter ecdysis in a second burst of protein synthetic activityat the time when there is replacement and growth of new musculartissue. The relation of these events with hormonal control ofgrowth is discussed.     

Protein synthesis in the embryos of Pinus thunbergii seed I. Influences of imbibition of seeds in the dark

The conditions and requirements of an in vitro protein-synthesizingsystem in the embryos of Pinus thunbergii seeds were studied.Even in the dry seed embryos, the ribosomes retained their syntheticcapacity. Even after imbibition in the dark, the ribosomes didnot show an increase in the activity of protein synthesis. Anincrease in the activity during dark imbibition was found inthe 100,000?g supernatant fraction. The activities of the cell-freesystems prepared from both embryos of dark-imbibed and dry seedswere dependent on the addition of poly U. This suggests thelack or inactivity of messenger RNA in these seed embryos. ¹ Present address: Faculty of Education, Utsunomiya University,Mine-machi, Utsunomiya 320, Japan. (Received July 19, 1976; )     

Accumulation of 70S Monoribosomes in Escherichia coli After Energy Source Shift-Down

When Escherichia coli is shifted from glucose-minimal to succinate-minimal medium, a transient inhibition of protein synthesis and a time-dependent redistribution of ribosomes from polysomes to 70S monosomes occurs. These processes are reversed by a shift-up with glucose. In a lysate made from a mixture of log-phase and down-shifted cells, the 70S monosomes are derived solely from the down-shifted cells and are therefore not produced by polysome breakage during preparation. This conclusion is supported by the absence of nascent proteins from the 70S peak. The monosomes are not dissociated by NaCl or by a crude ribosome dissociation factor, so they behave as "complexed" rather than "free" particles. When down-shifted cells are incubated with rifampin to block ribonucleic acid (RNA) synthesis, the 70S monosomes disappear with a half-life of 15 min. When glucose is also added this half-life decreases to 3 min. The 70S particles are stable in the presence of rifampin when chloramphenicol is added to block protein synthesis. We interpret these data to mean that the existence of the 70S monosomes depends on the continued synthesis of messenger RNA and their conversion to free ribosomes (which dissociate under our conditions) is a result of their participation in protein synthesis. Finally, a significant fraction of the RNA labeled during a brief pulse of (3)H-uracil is found associated with the 70S peak. These results are consistent with the hypothesis that the 70S monosomes are initiation complexes of single ribosomes and messenger RNA, which do not initiate polypeptide synthesis during a shift-down.     

The regulation of protein synthesis by translation control RNA

The mechanism by which translational control RNA (tcRNA) inhibits protein synthesis was investigated. In the presence of heme the inhibitory role of muscle tcRNA on hemoglobin synthesis was confirmed. Upon the addition of muscle tcRNA to a rabbit reticulocyte cell-free system the binding of [³²P]-globin mRNA to 40S ribosomal subunits and its subsequent incorporation into polysomes was inhibited. Furthermore, muscle tcRNA inhibits met-tRNA binding to polysomes and yet stimulates the formation of methionine-puromycin. These results suggest that muscle tcRNA blocks the binding of globin mRNA to ribosomes resulting in an abortive initiation complex that is, however, still capable of the methionine-puromycin reaction.     

Protein synthesis kinetics with ribosomes from temperature-sensitive mutants of Escherichia coli.

The kinetics of MS2 ribonucleic acid (RNA) directed protein synthesis have been investigated at seven temperatures between 30 and 47 degrees C by using ribosomes isolated from a wild type strain and seven temperature-sensitive mutants of Escherichia coli. The amount of MS2 coat protein formed at each temperature was determined by gel electrophoresis of the products formed with control ribosomes. With ribosomes from each of the mutant strains, the activation energy required to drive protein synthesis below the maximum temperature (up to 40 degrees C) was increased relative to the control (wild type) activity. Preincubation of the ribosomes at 44 degrees C revealed the kinetics of thermal inactivation, with ribosomes from each of the mutants having a half-life for inactivation less than that of the control ribosomes. A good correlation was observed between the relative activity of the different ribosomes at 44 degrees C and their relative rate of thermal inactivation. Mixing assays allowed the identification of a temperature-sensitive ribosomal subunit for each of the mutants. Defects in one or more of three specific steps in protein synthesis (messenger RNA binding, transfer RNA binding, transfer RNA binding, and subunit reassociation) were identified for the ribosomes from each mutant. The relationship between temperature sensitivity and protein synthesis in these strains is discussed.     

Polysomal Localization of R17 Bacteriophage-Specific Protein Synthesis

Synthesis of viral ribonucleic acid (RNA) polymerase, maturation protein, and coat protein in Escherichia coli infected with bacteriophage R17 occurs mainly on polysomes containing four or more ribosomes. The 30S ribosomal subunits through trimer-size polysomes, which are associated with all of the R17-specific proteins and are predominant in the infected cell, synthesize only coat protein. These structures may accumulate as products derived from larger polysomes as a result of failure in the release of nascent polypeptides after termination of chain growth. Appreciable amounts of viral coat protein remain attached to ribosomes and polysomes during R17 bacteriophage replication, supporting the hypothesis of the repressor role of this protein. The time course of synthesis of virus-specific proteins obtained from the polysomes of infected cells demonstrated regulated R17 messenger RNA translation consistent with the idea that coat protein is preferentially synthesized whereas the synthesis of noncoat proteins is suppressed.     

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 by long-lived messenger ribonucleic acid in bacteria

1. Experiments were performed to investigate two hypotheses about the function of long-lived messenger RNA in bacteria. After RNA synthesis had been stopped by the addition of actinomycin, continuing protein synthesis was used as a measure of persistent messenger RNA. 2. The hypothesis that messenger RNA responsible for the synthesis of membrane protein is exceptionally long-lived was tested in experiments with protoplasts of Bacillus megaterium. However, this messenger RNA proved to be of approximately average stability. 3. The hypothesis that long-lived messenger RNA is responsible for the synthesis of constitutive proteins was tested by comparing the synthesis of penicillinase in an inducible and a constitutive strain of Bacillus licheniformis. After the addition of actinomycin, penicillinase synthesis continued for far longer in the constitutive than in the inducible strain. This difference is attributed to a difference in stability of the penicillinase-messenger RNA in the two strains, which does not extend to all messenger RNA indiscriminately. 4. A model is tentatively proposed to account for the altered stability of messenger RNA in the constitutive mutant.     

Protein synthesis defects in temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins

The ribosomes from four temperature-sensitive mutants of Escherichia coli have been examined for defects in cell-free protein synthesis. The mutants examined had alterations in ribosomal proteins S10, S15, or L22 (two strains). Ribosomes from each mutant showed a reduced activity in the translation of phage MS2 RNA at 44 degrees C and were more rapidly inactivated by heating at this temperature compared to control ribosomes. Ribosomal subunits from three of the mutants demonstrated a partial or complete inability to reassociate at 44 degrees C. 70-S ribosomes from two strains showed a reducton in messenger RNA binding. tRNA binding to the 30 S subunit was reduced in the strains with altered 30-S proteins and binding to the 50 S subunit was affected in the mutants with a change in 50 S protein L22. The relation between ribosomal protein structure and function in protein synthesis in these mutants is discussed.     

Protein Synthesis in Cell-Free Systems: an Effect of Interferon

The activity of ribosome and cell-sap fractions from interferon-treated and control chick embryo fibroblasts was compared in mixed chick-mouse and purely chick cell-free systems capable of the synthesis of viral polypeptide(s) in response to viral ribonucleic acid (RNA). Interferon treatment of cells did not affect the intrinsic amino acid incorporation activity of these systems or their response to polyuridylic acid. With encephalomyocarditis (EMC) virus RNA as messenger, however, a fraction of the ribosomes from interferon-treated cells appeared less active than parallel controls. The results obtained with the corresponding cell-sap fractions were variable. Although competition between endogenous and added messengers cannot be excluded in these systems, a reduced level of translation of EMC RNA with interferon-treated cell ribosomes was also suggested by the results of analyses of tryptic digests of the products formed in response to the RNA. In addition, these analyses showed that this reduced activity must reflect a reduction in the rate or frequency of translation rather than a decrease in the length of the EMC RNA translated, for the same polypeptides were synthesized in response to the RNA with material from interferon-treated and control cells. Interferon added directly to the cell-free system was without effect. Although suggestive, these results do not provide definitive evidence for or against the hypothesis that virus protein synthesis is inhibited at the translational level in the interferon-treated cell. Possible alternative interpretations of the data are discussed.