A Messenger RNA for Tobacco Mosaic Virus Coat Protein in Infected Tobacco Mesophyll Protoplasts

Synthesis of tobacco mosaic virus (TMV)-specific low molecular weight component RNA (LMC) was investigated in relation to that of other TMV-related RNAs and proteins, and formation of progeny virus particles using synchronously infected tobacco mesophyll protoplasts. Timing of LMC synthesis was shown to be almost the same as, but somewhat earlier than that of TMV-RNA synthesis. In contrast, synthesis of TMV-specific double-stranded RNAs (replicative intermediate, RI and replicative form, RF) as well as a high molecular weight virus-induced protein (140 K protein) showed the maximum incorporation rate 4–6 h earlier than LMC synthesis. While, synthesis of coat protein and formation of progeny virus particles lagged behind LMC synthesis for 6–8 h. LMC occurring in polysomes was also investigated during the course of virus replication. The amount of produced coat protein calculated theoretically from the amount of LMC in polysomes of infected protoplasts was shown to be well agreed with the experimental results, indicating that LMC in polysomes is actively functioning as messenger for coat protien synthesis in vivo.     

Cellular site of synthesis of ribosomal proteins in yeast.

Looking for messenger RNA coding for yeast ribosomal protein, we devised a method to identify polysomes involved in ribosomal protein synthesis. Analysis of nascent protein elongated in vitro demonstrated that ribosomal proteins are synthesized both on membrane-associated and free polysomes.     

Protein synthesis in extracts from interferon-treated Mengo virus infected cells

We previously showed in intact L cells that interferon treatment did not modify the shut-off of cellular RNA and protein synthesis induced by infection with Mengo virus although viral replication is inhibited (1,2). We have also demonstrated that inhibition of host protein synthesis was not due to degradation of messengers since cellular mRNA could be extracted from interferon-treated infected cells and efficiently translated in a reticulocyte lysate(2). Cellular mRNA was not degraded although 2–5A was present as reported here. We prepared cell-free systems from such cells at a time when cellular shut-off was fully established. The undegraded messengers remained untranslated under cell-free protein synthesis conditions and almost no polysomes were detected. The decreased amount of [³⁵S]Met-tRNA-40S complex observed in these lysates might account for the inhibition of protein synthesis at the level of initiation.     

Both linked and unlinked mutations can alter the intracellular site of synthesis of exported proteins of Escherichia coli.

It previously has been demonstrated that synthesis of the periplasmic maltose-binding protein (MBP) and alkaline phosphatase (AP) of Eschericha coli predominantly occurs on membrane-bound polysomes. In this study, signal sequence alterations that adversely affect export of MBP and AP, resulting in their cytoplasmic accumulation as unprocessed precursors, were investigated to determine whether they have an effect on the intracellular site of synthesis of these proteins. Our findings indicate that export-defective MBP and AP are not synthesized or are synthesized in greatly reduced levels on membrane-bound polysomes. In some instances, a concomitant increase in the amount of these proteins synthesized on free polysomes was clearly discerned. We also determined the site of synthesis of MBP and AP in strains harboring mutations thought to alter the cellular secretion machinery. It was found that the presence of a prlA suppressor allele partially restored synthesis of export-defective MBP on membrane-bound polysomes. On the other hand, the absence of a functional SecA protein resulted in the synthesis of wild-type MBP and AP predominantly on free polysomes.     

Cellular mRNA translation is blocked at both initiation and elongation after infection by influenza virus or adenovirus.

During influenza virus infection, protein synthesis is maintained at high levels and a dramatic switch from cellular to viral protein synthesis occurs despite the presence of high levels of functional cellular mRNAs in the cytoplasm of infected cells (M. G. Katze and R. M. Krug, Mol. Cell. Biol. 4:2198-2206, 1984). To determine the step at which the block in cellular mRNA translation occurs, we compared the polysome association of several representative cellular mRNAs (actin, glyceraldehyde-3-phosphate dehydrogenase, and pHe7 mRNAs) in infected and uninfected HeLa cells. We showed that most of these cellular mRNAs remained polysome associated after influenza viral infection, indicating that the elongation of the proteins encoded by these cellular mRNAs was severely inhibited. Because the polysomes containing these cellular mRNAs did not increase in size but either remained the same size or decreased in size, the initiation step in cellular protein synthesis must also have been defective. Several control experiments established that the cellular mRNAs sedimenting in the polysome region of sucrose gradients were in fact associated with polyribosomes. Most definitively, puromycin treatment of infected cells caused the dissociation of polysomes and the release of cellular, as well as viral, mRNAs from the polysomes, indicating that the cellular mRNAs were associated with polysomes that were capable of forming at least a single peptide bond. A similar analysis was performed with HeLa cells infected by adenovirus, which also dramatically shuts down cellular protein synthesis. Again, it was found that most of the cellular mRNAs, which were translatable in reticulocyte extracts, remained associated with polysomes and that there was a combined initiation-elongation block to cellular protein synthesis. In cells infected by both adenovirus and influenza virus, influenza viral mRNAs were on larger polysomes than were several late adenoviral mRNAs with comparably sized coding regions. In addition, after influenza virus superinfection of cells infected by the adenovirus mutant dl331, a situation in which there is a limitation in the amount of functional initiation factor eIF-2 (M. G. Katze, B. M. Detjen, B. Safer, and R. M. Krug, Mol. Cell. Biol. 6:1741-1750, 1986), influenza viral mRNAs, but not late adenoviral mRNAs, were on polysomes. These results indicate that influenza viral mRNAs are better initiators of translation than are late adenoviral mRNAs.     

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.     

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.     

The formation and translational activity of polysomes from developing lupin seeds

The formation and in vitro translational activity of total, free and membrane-bound polysomes from various stages of developing cotyledons of yellow lupin seeds (Lupinus luteus L. cv. Iryd) has been investigated. The early stages of seed formation were characterized by a low level of polysomes that progressively increased. The main features of the cotyledons at the middle phase of development were full expansion growth and the highest amount of polysomes observed in all three poly so me fractions. In The final stages of emhryogenesis. the seed dehydration was accompanied by-gradual loss of all types of polysomes, at which the membrane-attached formations were degraded earlier than the free ones. By means of a wheat germ-derived cell-free system for protein synthesis, a correlation was demonstrated between cotyledon growth, polysome formation and their capacity for protein synthesis in vitro. As compared to the free polysomes, both the total and membrane-bound formations were more active in protein synthesis in vitro. Analysis of the translational products by means of immunoprecipitation and gel electrophoresis followed by fluorography showed that only membrane-bound polysomes produced polypeptides of higher molecular weight, including subunits of a legumin-like protein.     

Protein synthesis in homologous and heterologous cell-free systems from chick embryo connective tissues.

Two homologous systems for cell-free protein synthesis from chick embryo connective tissues are described. Both the skin polysomes and the wing-leg polysomes are active in collagen synthesis, but they have different requirements for optimum protein synthesis. Protein synthesis was not dependent on tissue-specific factors, since heterologous preparations of supernatant enzymes or initiation factors were able to stimulate maximum protein synthesis with each fraction of polysomes.     

Sequestration of pea reserve proteins by rough microsomes

Free polysomes, polysomes released from membranes, and rough microsomal vesicles isolated from developing cotyledons of Pisum sativum L. cv. Burpeeana were used to direct cell-free protein synthesis in a wheat germ system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the polypeptide products had molecular weights ranging from 12,000 to 74,000. Some of the polypeptides migrated during electrophoresis with the same mobility as polypeptides present in legumin and vicilin preparations. By the use of rabbit antibodies raised against pea reserve proteins it was established that polysomes released from membranes and rough microsomes directed the synthesis of polypeptides that were related to reserve proteins whereas free polysomes did not.     

Importance of cytomatrix-bound polysomes to synthesis of lysine-containing proteins in triticale germs under ABA treatment

The influence of exogenous abscisic acid (ABA) on the content of free polysomes (FP), membrane-bound polysomes (MBP), cytoskeleton-bound polysomes (CBP) and cytomatrix-bound polysomes (CMBP) in triticale germs as well as in vitro protein synthesis by these four polysomal fractions were studied. During translation, proteins were biotinylated for chemiluminescence detection. We have found that ABA changed both the content of FP, MBP, CMP and CMBP in germ tissue, and their subsequent translation activity. At 100 μM ABA, the content of FP and MBP was over fourfold lower compared to the control, whereas the amounts of CBP and CMBP were about two- and threefold higher, respectively. Moreover, the estimation of the share of polysomes in each ribosomal fraction (sub-units, monosomes, polysomes) showed that, at 100 μM ABA, cytomatrix-bound polysomes, which constituted 90% of polysomes, were the predominant class in ABA-treated germs while membrane-bound polysomes, which made up 82% of polysomes, dominated in the control. A high level of CMBP in ABA-treated tissues may indicate that this class of polysomes participates in ABA-induced synthesis of proteins. In turn, the inhibition of MBP under ABA-treatment is probably due to the delayed protein synthesis which takes place on these polysomes. We identified two lysine-containing proteins synthesized on both of the above classes of polysomes, whose synthesis was altered due to ABA application. Synthesis of a 47 kDa protein on MBP was inhibited, while synthesis of a 79 kDa protein on CMBP is strongly enhanced by ABA influence. The importance of these findings is discussed.     

PROTEIN SYNTHESIS IN PANCREAS OF FASTED PIGEONS

The regulation of protein synthesis in the pigeon has been studied by comparing the capability of cell-free amino acid incorporating systems of membrane-bound and membrane-free polysomes prepared from fasted and fed birds. New methods were developed for isolating polysomes since techniques used for other tissues did not provide quantitative recovery of polysomal RNA. The sucrose gradient profile of polysomes from pigeon pancreas showed a predominance of trisome species. Although initiation factors are present on polysomes, it was found that polysomes in cell-free systems would not initiate protein synthesis without exogenous initiation factors. This suggested the presence of an inhibitor or regulator of protein synthesis. These studies show that fasting resulted in: (a) decreased amounts of polysomes; (b) disaggregation of polysomes to monosomes; (c) decreased capability of polysomes to synthesize nascent peptides and to initiate additional synthesis, apparently not related to concentration of initiation factors.     

Translational control of protein synthesis after infection by vesicular stomatitis virus.

Four hours after infection of BHK cells by vesicular stomatitis virus (VSV), the rate of total protein synthesis was about 65% that of uninfected cells and synthesis of the 12 to 15 predominant cellular polypeptides was reduced to a level about 25% that of control cells. As determined by in vitro translation of isolated RNA and both one- and two-dimensional gel analyses of the products, all predominant cellular mRNA's remained intact and translatable after infection. The total amount of translatable mRNA per cell increased about threefold after infection; this additional mRNA directed synthesis of the five VSV structural proteins. To determine the subcellular localization of cellular and viral mRNA before and after infection, RNA from various sizes of polysomes and nonpolysomal ribonucleoproteins (RNPs) was isolated from infected and noninfected cells and translated in vitro. Over 80% of most predominant species of cellular mRNA was bound to polysomes in control cells, and over 60% was bound in infected cells. Only 2 of the 12 predominant species of translatable cellular mRNA's were localized to the RNP fraction, both in infected and in uninfected cells. The average size of polysomes translating individual cellular mRNA's was reduced about two- to threefold after infection. For example, in uninfected cells, actin (molecular weight 42,000) mRNA was found predominantly on polysomes with 12 ribosomes; after infection it was found on polysomes with five ribosomes, the same size of polysomes that were translating VSV N (molecular weight 52,000) and M (molecular weight 35,000) mRNA. We conclude that the inhibition of cellular protein synthesis after VSV infection is due, in large measure, to competition for ribosomes by a large excess of viral mRNA. The efficiency of initiation of translation on cellular and viral mRNA's is about the same in infected cells; cellular ribosomes are simply distributed among more mRNA's than are present in growing cells. About 20 to 30% of each of the predominant cellular and viral mRNA's were present in RNP particles in infected cells and were presumably inactive in protein synthesis. There was no preferential sequestration of cellular or viral mRNA's in RNPs after infection.     

A Messenger RNA for Tobacco Mosaic Virus Coat Protein in Infected Tobacco Mesophyll Protoplasts

The low molecular weight tobacco mosaic virus (TMV)-specific RNA component (LMC) was demonstrated in tobacco mesophyll protoplasts by polyacrylamide gel electrophoresis of ¹⁴C-uridine-labelled RNA from infected protoplasts. Free and membrane-bound polysomes were isolated from infected protoplasts, and RNA extracted from them was analyzed. TMV-specific RNA species including full-length viral RNA, its replicative intermediate, and LMC were found in both free and membrane-bound polysomes, but were present in free polysomes in much larger amounts. In particular, as much as 37 % of total LMC in protoplasts was found in free polysomes. Fractionation of polysomes by sedimentation in sucrose gradients showed that LMC is associated with small-sized polysomes (mono- to tetrasomes). Polysomes of this size class produced viral coat protein in a cell-free protein synthesizing system from rabbit reticulocytes. On the other hand, full-length TMV-RNA was associated predominantly with larger polysomes which produced in the cell-free system TMV-specific high molecular weight polypeptides but no coat protein. These results indicated that LMC, a subgenomic RNA of TMV, in fact functions in vivo as messenger RNA for viral coat protein, as has been postulated on the basis of in vitro studies.     

Translational alterations in maize leaves responding to pathogen infection, paraquat treatment, or heat shock : polysome dissociation and accumulation of a 57 kilodalton protein

Translational alterations occur in maize (Zea mays L.) leaves stressed by pathogen infection or herbicide paraquat treatment. These translational changes include: (a) dissociation of large polysomes to small polysomes, monosomes, and subunits; (b) a decreased rate of total protein synthesis; and (c) a reduced synthesis of several proteins by polysomes in vitro. The polysome dissociation was neither due to an extraction artifact nor to degradation of RNA by RNase. The protein patterns of polysomes isolated from leaves inoculated with Bipolaris maydis at 6 to 48 hours showed an increase in the intensity of a 57 kilodalton protein. When inoculated with less virulent pathogens, such as B. zeicola, Exserohilum turcicum, or Colletotrichum graminicola, the protein was accumulated in polysomes of leaves at 24 to 48 hours after inoculation. The 57 kilodalton protein was also accumulated in polysomes of maize leaves responding to heat shock or herbicide paraquat treatments. The purified 57 kilodalton protein reassociated with polysomes isolated from healthy leaves and inhibited polysomal translation in vitro. Since the 57 kilodalton protein is rapidly accumulated in maize polysomes in response to various biological and environmental stresses and may affect protein synthesis, it may be involved in translational regulation of maize leaves during stress response.     

Cycloheximide resistance in Chinese hamster cells. III. Characterization of cell-free protein synthesis by polysomes.

Two clones were selected for mass cultivation from 18 phenotypically stable CHM-resistant CHO clones. The polysomes isolated from these two clones were compared with CHO wildtype polysomes and rat liver polysomes in a cell-free protein synthesis system for their ability to incorporate amino acids. CHM had an inhibitory effect on the protein synthesis activity of CHO wildtype and rat liver polysomes, but had no effect on the polysomes obtained from either of the mutant CHO clones.     

Synthesis of two proteins in chloroplasts and mRNA distribution between thylakoids and stroma during the cell cycle of Chlamydomonas reinhardii

Chloroplasts contain thylakoid-bound and free ribosomes and polysomes. Whether binding of polysomes plays an immediate role in the regulation of chloroplast protein synthesis is not yet clear. In the present work, variations of protein synthesis and of mRNA content were measured not in greening, but in fully differentiated chloroplasts during the cell cycle of synchronized cultures of Chlamydomonas reinhardii. At different times of the vegetative cell cycle, the RNA was extracted from free and thylakoid-bound chloroplast polysomes and the partition of mRNAs between stroma and thylakoids was measured for two proteins, i.e. the 32-kDa herbicide-binding membrane protein and the soluble large subunit of the ribulose-1,5-bisphosphate carboxylase. At the same time the rates of synthesis of these two proteins were also determined. At 2 h after the onset of light, the content of both mRNAs in chloroplasts had doubled and 75-90% of each of these mRNAs were found to be bound to the thylakoids. The rate of protein synthesis, however, increased 10-fold, but reached its maximum only after about 6 h in the light. The differences in the time courses, in the stimulation of the rate of protein synthesis, and in the mRNA-binding to thylakoids point to a translational regulation of protein synthesis. Furthermore, since a very high proportion of polysomes were bound to thylakoids, containing mRNA for both a membrane and a soluble protein, this light-induced binding of polysomes to thylakoids seems to be an essential, but not the only, prerequisite for protein synthesis in chloroplasts.     

A polysome-membrane binding system from rat liver. I. Basic characterization of the binding system.

A simple reaction system was developed to examine the binding of polysomes to membranes of the endoplasmic reticulum and to investigate the fate of ribosomes and nascent chains during protein synthesis in vitro. The system conssited of Sephadex G-25 treated post-mitochondrial fraction prepared from rat liver (Sephadex-PM) as a source of membranes, and radioactive free polysomes prepared from another rat liver. The following results were obtained. 1. Nascent chains on free polysomes labeled in vivo were transferred to membranes in vitro. The process required protein synthesis. 2. This reaction occurred in two steps: a) Binding of the free polysomes to membranes in the absence of protein synthesis. b) Release of ribosomes, leaving nascent chains on the membranes, requiring protein syntehsis. 3. A portion of the ribosomes found on membranes in vivi (membrane-bound ribosomes) was also released from the membranes during incubation in vitro, leaving their nascent chains on the membranes. The significance of the transfer of nascent chains from free polysomes to membranes in vitro is discussed in the light of known polysome-membrane interaction in vivo.     

Synthesis of S100 Protein on Free and Membrane-Bound Polysomes of the Rabbit Brain

Abstract: Free and membrane-bound polysomes were isolated from the cerebral hemispheres and cerebellum of the young adult rabbit. The two polysomal populations were translated in an mRNA-dependent cell-free system derived from rabbit reticulocytes. Analysis of the [³⁵S]methionine-labeled translation products on two-dimensional polyacrylamide gels indicated an efficient separation of the two classes of brain polysomes. The relative synthesis of S100 protein by free and membrane- bound polysomes was determined by direct immuno-precipitation of the cell-free translation products in the presence of detergents to reduce nonspecific trapping. Synthesis of S100 protein was found to be twofold greater on membrane-bound polysomes compared with free polysomes isolated from either the cerebral hemispheres or the cerebellum. In addition, the proportion of poly- (A+)mRNA coding for SlOO protein was also twofold greater in membrane-bound polysomes compared with free polysomes isolated from the cerebral hemispheres. These results indicate that the cytoplasmic S100 protein is synthesized predominantly on membrane-bound polysomes in the rabbit brain. We suggest that the nascent S100 polypeptide chain translation complex is attached to the rough endoplasmic reticulum by an ionic interaction involving a sequence of 13 basic amino acids in S100 protein.