On the accessibility and selection of the initiator site of mRNA in protein synthesis

The specificity of the cell-free system of Escherichia coli for mRNA was examined, and the “accessibility” of some natural and synthetic RNAs to the ribosomes was determined by measurement of AcPhe-tRNA and fMet-tRNA binding, AcPhe-puromycin and fMet-puromycin formation, and polypeptide synthesis. The E. coli system effectively initiates the translation of various synthetic RNAs with AcPhe-tRNA or fMet-tRNA under conditions optimal for the translation of viral RNA. Poly(A,G,U) is accessible to the ribosomes according to all of the above criteria. Poly(A,C,G,U), 23 S rRNA, R17 RNA, and MS2 RNA, on the other hand, show limited accessibility when tested for initiator tRNA binding, or for AcPhe-puromycin and fMet-puromycin formation. MS2 and R17 RNA, but not poly(A,C,G,U) and 23 S rRNA, show accessibility when measured by polypeptide synthesis. The results suggest that, except at initiator sites of natural mRNA, an RNA containing about equal amounts of all four bases is inaccessible to E. coli ribosomes for polypeptide synthesis. Rate constants obtained for fMet-tRNA binding with MS2 RNA, poly(A,G,U), and poly(C,G,U) indicate that the ribosomes do not have any special affinity for the viral RNA. Thus, the selection of the initiator site in protein synthesis may be critically determined more by the accessibility of the initiator codon than by ribosomal recognition of the site.     

Synthesis of Chloroplast Proteins during Germination and Early Development of Cucumber

Cell-free protein synthesizing systems have been used to study the developmental changes in the synthesis of chloroplast proteins in the cotyledons of cucumber seedlings grown in the light or in the dark. Escherichia coli and wheat germ in vitro protein synthesizing systems have been used to assay the changes in the levels of the mRNA's coding for ribulose 1,5-bisphosphate carboxylase (RuBPCase). The large subunit of cucumber RuBPCase has been identified among the translation products of the E. coli system. The wheat germ system translates the cucumber mRNA coding for the small subunit of RuBPCase to produce a 25,000 molecular weight precursor polypeptide. Plastids isolated from light-grown cotyledons were used to study developmental changes in their capacity to synthesize protein. The data obtained indicate that in the light there is an initial 48-hour period of accumulation of the mRNA's coding for the large and small subunits of RuBPCase, coupled with an increase in the capacity of the isolated plastids to synthesize protein. This is followed by a decline. This decline is not reflected in the accumulation of RuBPCase in the cotyledons which remains constant over the period of study.     

Protein synthesis in chloroplasts: V. Translation of messenger RNA for the large subunit of fraction I protein in a heterologous cell-free system

The addition of spinach chloroplast total RNA to cell-free extracts from Escherichia coli stimulates amino acid incorporation into protein. The products were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and were qualitatively and quantitatively similar to those synthesized in intact isolated chloroplasts. There are two major discrete products of both systems with molecular weights of 52,000 and 35,000. The [³⁵S]methionine-containing chymotryptic peptides of the 52,000 M_r polypeptide synthesized in the E. coli cell-free system have been compared with those of fraction I protein large subunit labelled with [³⁵S]methionine in vivo. From the close similarity in chromatographic properties of the peptides of the two polypeptides, we conclude that the 52,000 M_r product of chloroplast RNA-directed protein synthesis in E. coli extracts is the large subunit of fraction I protein.     

Cell-free translation of chloroplast RNAs in the wheat germ system

Spinach, tobacco and Euglena chloroplast RNAs (cp RNA) can be successfully translated in the wheat germ cell-free system. The in vitro translation products obtained from spinach cp RNA in the wheat germ and in the Escherichia coli system are similar to each other and to that of in organello synthesis, if analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Translation of mixtures of chloroplast and total RNA of leaves reveals that under conditions of mRNA competition the cytoplasmic type of RNA is preferentially translated in the wheat germ system.     

Electrophoretic transfer as a technique for the detection and identification of plant amylolytic enzymes in polyacrylamide gels

Polyadenylated RNA was isolated from leaves and seeds of a C₃ plant (Triticum aestivum L. cv Cheyenne, CI 8885) and from a C₄ plant (Zea mays L. cv Golden bantam). Each polyadenylated RNA preparation was translated in vitro with micrococcal nuclease-treated reticulocyte lysate. When the in vitro translation products were probed with antibodies to pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1), two sizes of polypeptide were identified. A 110 kilodalton polypeptide was found in the in vitro translation products of mRNA isolated exclusively from leaves of both wheat and maize. A 94 kilodalton polypeptide, similar to the PPDK polypeptide which can be extracted after in vivo synthesis in maize and wheat leaves and seeds, was found in the in vitro translation products obtained from wheat seeds and maize kernels.

These results indicate that the mRNAs for PPDK polypeptides are organ-specific in both a C₄ and a C₃ plant. Hague et al. (1983 Nucleic Acids Res 11: 4853-4865) proposed that the larger size polypeptide of the in vitro translation polypeptide from maize leaf RNA contains a `transit sequence' which permits entry into the chloroplasts of a polypeptide synthesized in vivo in maize leaf cell cytoplasm. It appears that in wheat leaves also the transit of synthesized PPDK polypeptide through an intracellular membrane may be required, while such a transit sequence seems not to be required within cells of wheat and maize seeds.

     

CsdA, a cold-shock RNA helicase from Escherichia coli, is involved in the biogenesis of 50S ribosomal subunit

CsdA, a DEAD-box protein from Escherichia coli, has been proposed to participate in a variety of processes, such as translation initiation, gene regulation after cold-shock, mRNA decay and biogenesis of the small ribosomal subunit. Whether the protein really plays a direct role in these multiple processes is however, not clear. Here, we show that CsdA is involved in the biogenesis of the large rather than the small ribosomal subunit. Deletion of the csdA gene leads to a deficit in free 50S subunits at low temperatures and to the accumulation of a new particle sedimenting around 40S. Analysis of the RNA and protein contents of this particle indicates that it corresponds to a mis-assembled large subunit. Sucrose gradient fractionation shows that in wild-type cells CsdA associates mainly with a pre50S particle. Presumably the RNA helicase activity of CsdA permits a structural rearrangement during 50S biogenesis at low temperature. We showed previously that SrmB, another DEAD-box RNA helicase, is also involved in 50S assembly in E.coli. Our results suggest that CsdA is required at a later step than SrmB. However, over-expression of CsdA corrects the ribosome defect of the srmB-deleted strain, indicating that some functional overlap exists between the two proteins.     

Quantitative and qualitative variation in the mRNA for carboxylesterases in insecticide-susceptible and resistant Myzus persicae (Sulz)

RNA was extracted from two insecticide-resistant clones and one susceptible clone of M. persicae. The resistant clones each produced large amounts of one of two closely related carboxylesterases, the enzymes responsible for cross-resistance to a wide range of insecticides. After purification by affinity chromatography on oligo(dT) cellulose, the mRNA was translated into protein in a rabbit reticulocyte lysate system with [l-³⁵S]methionine. The resultant radiolabelled esterases were immunoprecipitated from the products with IgG prepared from an antiserum to one form of the enzyme, but cross-reacting with both. The bound enzyme was extracted by affinity chromatography on protein A sepharose, and characterized alongside the total radiolabelled proteins by SDS electrophoresis and fluorography. The translation products of the two resistant clones each contained large amounts of an immunoprecipitable protein. However, no such protein was detected in the translation products of the mRNA from susceptible aphids showing that resistant aphids produce much more of the mRNA encoding the enzymes responsible for resistance. It was also shown that the enzymes from the two resistant clones had primary structures differing from each other by 1 kDa. In addition, the nascent forms of both enzymes differed from their native forms by 8 kDa and glycosylation was shown to be responsible for this post-translational modification. The likely genetic basis of the changes in mRNA is discussed and related to the karyotype of the resistant clones.     

Synthesis of the cyanogenic beta-glucosidase, linamarase, in white clover

The beta-glucosidase, linamarase, which specifically hydrolyzes cyanogenic substrates, linamarin and lotaustralin, in white clover, is synthesized in the early stages of leaf and seedling development in genetically competent plants. Plants, from natural populations, possessing at least one Li allele synthesize linamarase but plants with only li alleles do not, nor do they produce inactive but antigenically related linamarase. Linamarase is known to be a mannosyl glycoprotein, which in its active form is a dimer, with a subunit size of 62,000 Mr. We demonstrate that the antibiotic tunicamycin, which prevents N-acetyl-asparagine linked glycosylation, reduces in vivo synthesis of linarmarase. In vitro translation of mRNA from a Li Li plant yields a 59,000 Mr immunoprecipitated linamarase polypeptide which is modified to a 62,000 Mr product by the addition of dog pancreas microsomes. No anti-linamarase immunoprecipitable product is obtained from the in vitro translation products of mRNA from a li li plant.     

Two ribosome binding sites from the gene 0.3 messenger RNA of bacteriophage T7

Ribosome-protected regions have been isolated and analyzed from the bacteriophage T7 gene 0.3 mRNA labeled in vivo. Two discrete sites which are nearly equally protected by ribosomes are obtained from what was previously assumed to be a monocistronic message. Use of appropriate T7 deletion mutant RNAs has allowed mapping of both ribosome-recognized regions. Site a is positioned very close to the 5′ terminus of the mRNA and is apparently the initiator region for the major gene 0.3 protein, which acts to overcome the host DNA restriction system. Site b is located within several hundred nucleotides of the 3′ end of the RNA and probably initiates synthesis of a small polypeptide of unknown function. Both ribosome binding sites exhibit features common to other initiator regions from Escherichia coli and bacteriophage mRNAs. The proximity of site a to the RNase III cleavage site at the left end of gene 0.3 may explain why processing by RNase III is required for efficient translation of the major gene 0.3 protein.     

Product of in vitro translation of the Rous sarcoma virus src gene has protein kinase activity.

In vitro translation of Rous sarcoma virus virion RNA resulted in the synthesis of a protein kinase which, when immunoprecipitated with antitumor serum, phosphorylated the immunoglobulin heavy chain. Even though in vitro translation of virion RNA resulted in the synthesis of a number of polypeptides which were recognized by antitumor serum, control experiments demonstrated that an immunoprecipitable protein kinase activity was found only when an immunoprecipitable p60src, the polypeptide product of the src gene, was synthesized. A protein kinase with similar properties was therefore intimately associated with p60src which was synthesized in vitro in the reticulocyte lysate, just as it is with p60src which is obtained from transformed chick and mammalian cells. It is therefore highly unlikely that this association is artifactual. ts NY68 is a mutant of Rous sarcoma virus which is able to transform cells at 36 but not at 41 degrees C. In vitro translation of ts NY68 virion RNA at 30 degrees C resulted in efficient synthesis of immunoprecipitable p60src, but very inefficient synthesis of an immunoprecipitable protein kinase. The p60src obtained by in vitro translation of wild-type virion RNA was more than 20-fold more active as a protein kinase than was that obtained from ts NY68 RNA. The correlation in the case of ts NY68 of a deficiency in protein kinase activity with an inability to transform cells at high temperature suggests that the protein kinase activity associated with p60src is indeed critical to cellular transformation.     

Inhibition of phytoalexin biosynthesis in elicitor-treated tobacco cell-suspension cultures by calcium/calmodulin antagonists

A role for calcium/calcium-binding proteins in a mechanism of signaling elicitor-inducible phytoalexin biosynthesis was investigated. Two classes of calcium/calmodulin antagonists, phenothiazines and naphthalenesulfonamides, inhibited sesquiterpene phytoalexin accumulation in tobacco (Nicotiana tabacum) cell-suspension cultures when added 1 h before elicitor. The antagonists also inhibited the induction of sesquiterpene cyclase enzyme activity, a key regulatory enzyme for sesquiterpene biosynthesis. The antagonists suppressed the induction of sesquiterpene cyclase only if added before or simultaneously with elicitor. Additionally, the antagonists inhibited (a) accumulation of the cyclase protein as measured in immunoblots; (b) the in vivo synthesis rate of the cyclase protein, measured as the incorporation of [³⁵S]methionine into immunoprecipitable cyclase protein; and (c) the cyclase mRNA translational activity, measured as the incorporation of [³⁵S]methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of RNA isolated from antagonist-treated, elicitor-induced cells. In contrast, elicitor-inducible phenylalanine ammonia lyase enzyme activity, the level of the enzyme protein, the in vivo synthesis rate, and the mRNA translational activity were not affected by any of the antagonist treatments. Uptake and incorporation of [³⁵S]methionine into total cellular proteins and total in vitro translation products were also not indiscriminately altered by the antagonist treatments. The current results suggest that calcium and/or calmodulin-like proteins may be elements of a signal transduction pathway mediating elicitor-induced accumulation of phytoalexins in tobacco.     

Competition between bacteriophage f2 RNA and bacteriophage T4 messenger RNA

In an $\text{Escherichia coli}$ cell-free protein synthesis assay, mRNA isolated from cells late after infection by phage T4 out-competes bacteriophage f2 RNA. Addition of a saturating or subsaturating amount of T4 mRNA inhibits translation of f2 RNA, while even an excess of f2 RNA has no effect on translation of T4 mRNA. Peptide mapping of reaction products labeled with formyl-[³⁵S]-methionyl-tRNA was used to quantitate f2 and T4 protein products synthesized in the same reaction. We suggest that messenger RNA competition might be one mechanism by which T4 superinfection of cells infected with phage f2 blocks translation of f2 RNA and possibly host mRNA.     

Translation of bacteriophage R17 and Qbeta RNA in a mammalian cell-free system

The polycistronic RNAs from both bacteriophage R17 and Qβ are translated in a mammalian cell-free system of purified and partially purified components. The requirement of one of the partially purified initiation factors (IF-E₃ from rabbit reticulocytes) for the phage RNA translation is strikingly different from that for rabbit globin messenger RNA translation. The phage RNA-directed products are characterized by acrylamide gel electrophoresis and compared with those synthesized in an Escherichia coli cell-free system. There is good agreement between the respective coat proteins and the presumptive synthetase proteins. R17 RNA directs the synthesis of two additional defined polypeptides. However, their possible relationship with the A-protein cistron has not yet been investigated. The RNA from the amB2 mutant of R17, which carries an amber triplet at position 6 in the coat protein cistron, directs the synthesis of the same polypeptides as the wild-type RNA with the exception of the coat protein which is completely abolished. This identifies the product made with wild-type RNA as coat protein and provides a direct in vitro assay for the suppression of nonsense mutations in eukaryotic cells.     

A Nicotiana tabacum mRNA encoding a 69-kDa glycoprotein occurring abundantly in pollen tubes is transcribed but not translated during pollen development in the anthers

Regulation of expression of a 69-kDa glycoprotein which occurs abundantly in tobacco (Nicotiana tabacum L.) pollen tubes but is absent in ungerminated pollen has been studied in vitro by means of a coupled translation/glycosylation system with RNA isolated from various stages of pollen development. Pollen mRNA could be translated in a rabbit reticulocyte lysate and the products glycosylated with canine pancreatic microsomal membranes. The electrophoretic pattern of translation products obtained with pollen-tube RNA showed a prominent polypeptide with an apparent molecular mass of 58 kDa. In the presence of the canine pancreatic microsomal membranes this polypeptide was glycosylated, producing the 69-kDa glycoprotein. The presence of mRNA encoding the 58-kDa precursor polypeptide was also demonstrated in ungerminated pollen and in young mid-binucleate pollen isolated from anthers. Initiation of synthesis of the 69-kDa glycoprotein at the onset of pollen germination thus occurs through unmasking of the mRNA transcribed during pollen differentiation and stored during pollen maturation and dormancy in an inactive state.Abbreviation pI isoelectric point