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Presents a list of research papers to be published in the upcoming issues of the "European Journal of Biochemistry." "Extraction and Translation of Collagen mRNA From Fetal Calf Skin," by R. Kaufman, A. Belayew, B. Nusgens, and C. M. Lapie&rgrave;e; "Messenger RNA For Ribosomal Proteins in Xenopus laevis Oocytes," by P. Pierandrei-Amaldi and E. Beccari; "Purification and Characterization of Esterase 2B of the House Mouse, Mus musculus," by U. Lexow, A. Ronai, and O. von Deimling.

UTP-modulated attenuation of transcription is involved in regulating the synthesis of pyrimidine nucleotides in Escherichia coli. Thus, expression of two genes, pyrBI and pyrE, was shown to be under this type of control. The genes encode the two subunits of aspartate transcarbamylase and orotate phosphoribosyltransferase respectively. The levels of these enzymes are inversely correlated with the intracellular concentration of UTP. Modulation of attenuation seems to be a consequence of the effect of UTP concentration on the mRNA chain growth rate. Reducing the UTP pool retards RNA polymerase movement. Mechanistically this will couple the ribosomes translating a leader peptide gene more tightly to the elongating RNA polymerase. The ribosomes will then be more prone to prevent the folding of the mRNA chains into terminating hairpin structures when RNA polymerase is at the attenuator and has to decide whether transcription should terminate or continue into the structural genes. This paper described a study of pyrBI and pyrE gene regulation in cells where the ribosomes move slowly as a result of mutation in rpsL. It appears that expression of the two genes is hyper-regulated by the UTP pool in this type of cells. Furthermore, the attenuator model can only account for the results if it is assumed that UTP-concentration dependent pausing of transcription occurs in vivo in the two pry gene leaders such that RNA polymerase waits for the coupled ribosomes before transcribing into the attenuator regions. [ABSTRACT FROM AUTHOR]

The confronmation in solution of duck and rabbit globin mRNA, and of the duck mRNA in the mRNA · protein particle, has been investigated by optical methods and also by the use of the dye ethidium bromide which becomes highly flourescent when intercalated into the double-stranded regions of a nucleic acid. On the basis of the properties of this dye and on the ability of homopolyribonucleotides to form double-stranded structures we have, in addition, developed a simple and sensitive assay for the detection and quantitisation of sequences rich in a particular residue that may be present in an RNA chain. In solutio, 45 to 60% of the nucleotides of duck globin mRNA were found to be in bihelical regions. A similar degree of secondary structure was found in rabbit globin mRNA (this paper), as well as in calf lens mRNA and mRNAs from ewe mammary gland (other results). All samples of globin mRNA examined in this work contained a sequence of poly(A), which has poly(U) binding properties similar to that of synthetic poly(A): no specific interaction between the poly(A) sequence and the rest of the molecules can be detected. The fraction of adenosine residues within these poly(A) segments represents 4% in rabbit mRNA and 8 to 9% in duck mRNA. An additional adenosine-rich segment interspersed with guanosine and possibly residues, was also detected in one duck mRNA sample. The RNA in the duck mRNA · protein particle is also highly structured. The melting profile in the range of 20 to 65° C is quite similar to that of free mRNA and the ability of ethidium bromide to intercalate is reduced to the extent of 70%. Yet the dichroïc spectra of free and bound mRNA are significantly distinct. These data suggest that free and protein-bound mRNA may have a very similar degree of secondary structure but with distinct detailed conformation in bihelical regions (change in base tilting for example). Direct evidence has been obtained that proteins stick to the poly(A) segment in the particle since the fraction of adenosine residues detectable by our poly(A) titration procedure is reduced to 50% of that observed in the free mRNA. [ABSTRACT FROM AUTHOR]

A technique which releases ribosomes from membranes without the use of detergent described in the preceding paper is shown to allow the separation of two distinct pools of poly(A)-containing RNA in microsomes. Thus, using preparations from mouse myeloma tissue-culture cells, essentially 100 % release of immunoglobulin mRNA occurs under conditions in which only 50-60 % of the total microsomal poly(A)-containing RNA is released. The RNA which is not released is principally mitochondrial ribosomal RNA and the method thus provides a good preparative procedure for this fraction. Messenger ribonucleoprotein particles involved in immunoglobulin biosynthesis have been prepared from the released material. The heavy-chain mRNA-containing particle sediments in a neutral sucrose gradient at about 22 S, and that containing the light-chain mRNA at about 18 S. The ribosome-release technique has been used for the rapid preparation of light-chain mRNA in good yield and at over 60 % purity after a single sucrose gradient centrifugation. Further purification has been achieved by polyacrylamide gel electrophoresis. Using [32P]RNA, the contaminants can be estimated directly at the chemical leveI using a fingerprint assay. [ABSTRACT FROM AUTHOR]

The regulation of ribosomal proteins (r-proteins) and their mRNAs (rp-mRNAs) was studied in the L6 myoblast, a mammalian cell line which can undergo myogenesis. Upon terminal differentiation, the rate of accumulation of mature ribosomes dropped to approximately 25% of the rate found in undifferentiated myoblasts. Despite the drop in the rate of ribosome accumulation and the rate of rRNA synthesis following terminal differentiation, the rate of r-protein synthesis remained constant. The excess r-protein synthesized in myotubes was quickly degraded. The levels of rp-mRNAs were assessed before and after differentiation. Over 90% of the rp-mRNAs were found on polysomes in both myoblasts and myotubes and represented similar fractions of total poly(A)-rich mRNA. The half-lives of the rp-mRNAs averaged approximately 11 h in both myoblasts and myotubes. In vitro nuclear transcription measurements of a representative rp-mRNA (L32 mRNA) revealed that following differentiation, its rate of synthesis relative to the overall transcription rate dropped by approximately 26% in myotubes while the rate of transcription of rRNA dropped by approximately 77%. These results indicate that the coordination of r-protein and rRNA synthesis observed in myoblasts was uncoupled in myotubes at the level of transcription. [ABSTRACT FROM AUTHOR]

The study of polysomal formation in the ts-136 thermo-sensitive yeast mutant indicates that ribosomes are assembled with an mRNA, which is in a structure tightly bound to membranes, and are then released into the soluble cytoplasmic fraction. This has been observed by studying the recruitment of ribosomes when glucose is added to glucose-starved cells and when transport of mRNA is permitted by shifting the mutant to the permissive temperature. A soluble cytoplasm and a fraction becoming soluble after sodium deoxycholate treatment of a rapidly sedimenting structure have been characterized. The former contains the majority of polysomes, free 80-S monomers and almost all of the ribosomal subunits. The latter fraction is composed of bound 80-S monomers and polysomes, but lacks ribosomal subunits. Treatment of the rapidly sedimenting structure with pancreatic ribonuclease produces the release of 80-S monomers, with EDTA the release of an equal proportion of both ribosomal subunits, and with sodium deoxycholate the release of 80-S monomers and polysomes. These findings are consistent with the assumption that bound ribosomes are assembled with an mRNA which is tightly bound to this rapidly sedimenting structure, presumably membranes. From the operational viewpoint this fraction is called the "membrane." During the process of polysomal formation ribosomes are recruited more rapidly in the "membranes" than in the soluble cytoplasm. Since "membranes" do not accumulate polysomes and contain only a small fraction of the total amount of ribosomes, the result is consistent with the assumption that either there is a higher turnover of bound versus free polysomes or bound polysomes are the precursors of free polysomes. The latter assumption is more likely since we have shown previously (a) that in yeast, transport is coupled with the translation of bound mRNA, (b)that this mRNA is tightly bound to a structure which sediments very rapidly and becomes soluble only after sodium deoxycholate treatment, and (c) when cycloheximide is added during the recruitment of ribosomes there is accumulation of membrane-bound ribosomes. [ABSTRACT FROM AUTHOR]

Both rRNA and protein components of Escherichia coli ribosomes have been specifically labelled without any important loss of activity for polyphenylalanine synthesis in the cell-free system. Three adenylic residues per 70-S ribosome were labelled by an anthracenyl derivative and the ribosomal proteins by dansylation of four amino acid residues per 70-S particle. The association between the two complementary subparticles, the formation of the binary and ternary complexes in the presence of mRNA and tRNA were investigated using the concomitant measuring of fluoroscence intensity and polarisation of the two kinds of labels. The formation of these complexes involves the rRNA and the ribosomal protein components of the 30-S and 50-S subparticles directly and/or indirectly. The mobility of the messenger bound to the 70-S ribosome in the presence of tRNA is indicated by use of a polyadenylic acid labelled with the same marker ss that for rRNA. Reversible conformational changes are observed during acidic-alkaline titrations and melting curves of fluorescent ribosomal particles. The irreversible denaturation occurs at the same pH and melting temperature for the rRNA and the ribosomal components: The results are interpreted in terms of highly specific interactions between the rRNA and the proteins, slight modification in the strength and/or nature of these interactions leading to a conformational change of the whole ribosomal particle. [ABSTRACT FROM AUTHOR]

Affinity labeling of human placental 80S ribosomes with mRNA analogs of up to 12 uridyl residues, i.e. alkylating derivatives of oligouridylates bearing either 4-(N-2-chloroethyl-N-methyl-amino) benzylmethylphosphamide group at the 5'-termini or 2',3'-O-[4-(N-2-chloroethyl-N-methyl-amino)] benzylidene residue attached to the 3'-termini. in the presence of cognate Phe-tRNAPhe has been investigated. All the mRNA analogs modified only the 40S subunit. The fraction of 18S rRNA modified by the mRNA analogs with the alkylating group at the 5'-end decreased dramatically with extension of the reagent oligouridylate moiety. Nucleotides of 18S rRNA alkylated with the mRNA analogs were determined using a reverse transcription technique. For the mRNA analogs with the alkylating groups at the 3'-termini, G1702 and G1763/G1764 were identified as the cross-linking sites. The intensities of the bands corresponding to reverse transcriptase stops depended on the length of the reagent oligouridylate moieties. Cross-linking sites of the mRNA analogs with the alkylating group at the 5'-termini on 18S rRNA were A1023, C1026, C1057 and A1058 for the (pU)3 and (pU)4 derivatives and a single nucleotide C1057 for the (pU)6 one. Ribosomal protein S26 was found as the main target of modification with the same derivatives of (pU)6 and (pU)12. [ABSTRACT FROM AUTHOR]

Prosomes were first described as being mRNA-associated RNP (ribonucleoprotein) particles and subcomponents of repressed mRNPs (messenger ribonucleoprotein). We show here that prosomes isolated from translationally inactive mRNP have a protease activity identical to that described by others for the multicatalytic proteinase complex (MCP, ‘proteasome’). By RNase or non-ionic detergent treatment, the MCP activity associated with repressed non-globin mRNP from avian erythroblasts, sedimenting at 35 S, could be quantitatively shifted on sucrose gradients to the 19-S sedimentation zone characteristic of prosomes, which were identified by monoclonal antibodies. The presence of small R NA in the enzymatic complex was shown by immunoprecipitation of the protease activity out of dissociated mRNP using a mixture of anti-prosome monoclonal antibodies; a set of small RNAs 80–120 nucleotides long was isolated from the immunoprecipitate. Furthermore, on CsCl gradients, colocalisation of the MCP activity with prosomal proteins and prosomal RNA was found, and no difference in the prosomal RNA pattern was observed whether the particles were fixed or not prior to centrifugation. These data indicate that the MCP activity is a property of prosomes, shown to be in part RNP and subcomplexes of in vivo untranslated mRNP. A hypothesis for the role of the prosome-MCP particles in maintaining homeostasis of specific protein levels is proposed. [ABSTRACT FROM AUTHOR]

We have used site-directed mutagenesis to determine whether the structural context surrounding the AUG triplet influences its ability to be selected an an initiation codon by the eukaryotic preinitiation complex. AUG triplets were introduced in a loop and stem structure naturally occurring at the midpoint of the 129-nucleotides-long 5’-untranslated region of the porcine proopiomelanocortin mRNA; one AUG triplet was inserted in the loop while another was inserted in the stem of the hairpin structure. The proopiomelanocortin cDNA and the mutant cDNAs were inserted downstream from the early promoter of an expression vector derived from simian virus 40 (SV40) and transfected into monkey kidney COS-1 cells. Analysis of the proopiomelanocortin-related peptides presents in the culture medium 72 h after transfection revealed that both mutant cDNAs direct the synthesis of more proopiomelanocortin than the non-mutant cDNA. The increased translational efficiency observed with both mutants is probably due to the decreased secondary structures of the shortened 5’-untranslated region. In addition, comparison of the two mutants indicates that the mutant mRNA with the AUG triplet inserted in the loop region of the hairpin structure directs the synthesis of approximately 75% more proopiomelanocortin than the mutant mRNA with the AUG triplet inserted in the stem region of the same hairpin structure, supporting a role for the structural context in the efficiency of translational initiation. [ABSTRACT FROM AUTHOR]

In the leaves of rye (Secale cereale L.), control mechanisms acting at multiple molecular levels contribute to a coordinate expression of the subunit polypeptides of ribulose-1,5-bisphosphate carboxylase. The relevance and hierarchy of the different control steps were evaluated by comparing the time courses of changes in levels of translatable mRNA, rates of in vivo amino acid incorporation, and the turnover of subunit polypeptides after selective interference with translation at either cytoplasmic 80S ribosomes, or at the 70S ribosomes of the chloroplast, by compartment-specific inhibitors, or by the use of 70S-ribosome-deficient leaves. The latter were generated by growing the plants at a non-permissive elevated temperature of 32°C. The rates of synthesis of the two ribulose-1,5-bisphosphate carboxylase subunits were most rapidly adapted to each other by translational controls. Within 0.5–2.5 h after selective inhibition of the synthesis of either subunit, that of the other subunit made in the unaffected compartment also declined by more than 90% without any marked change in its mRNA. After prolonged inhibition (24 h) of either cytoplasmic or chloroplast protein synthesis, the levels of mRNAs for both subunits were greatly diminished. In rye, the mRNA levels for both subunits changed under all experimental conditions tested in a closely parallel manner and appeared to be always maintained in a balanced, fairly constant ratio by strong coordinate controls. Even 70S-ribosome-deficient leaves contained mRNAs for both the small and the large subunits, although only in small amounts. The mRNAs for both subunits were also markedly further decreased in 70S-ribosome-deficient leaves after application of an inhibitor of cytoplasmic translation, MDMP [2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide], suggesting that the suppression of the large subunit mRNA in the plastids was not mediated through feedback effects of accumulating unassembled large subunits. Coordinate controls at both the mRNA and the translational level require a bidirectional exchange of regulatory signals between chloroplast and cytoplasm. However, these controls were not absolutely restrictive and allowed low rates of uncoupled synthesis of either large or small subunits. Large subunits made in the presence of MDMP were stable over 24 h. However, unassembled small subunits synthesized in 70S-ribosome-deficient leaves were degraded with a half-time of 10.5 h, in contrast to their behavior after integration into the holoprotein in normal leaves, where no turnover was detected. The protcolytic removal of surplus free small subunits is regarded as a final post-ranslational fine-tuning step to establish a balanced subunit stoichiometry in leaves. [ABSTRACT FROM AUTHOR]

The misincorporation of cysteine (codon: UGU/C) into twelve ribosomal proteins devoid of cysteine has been studied. Although it is generally assumed that cysteine is misincorporated at arginine and tryptophan residues (codons: CGU/U and UGG respectively), our results are consistent with the idea that cysteine is also mis-incorporated at phenylalanine residues (codon: UUU/C) through a second-position C : U mismatch. Cysteine was found in ribosomal proteins L29, L32/L33 and SW, under conditions where only its misincorporation at neutral residues was measured. Since these proteins contain no tryptophan, the data imply that cysteine has replaced a neutral amino acid other than tryptophan. Because there was a statistically significant correlation between the total level of cysteine in the twelve proteins under study and their content of phenylalanine and arginine residues, we conclude that there is a likelihood of cysteine misincorporation at phenylalanine residues, in addition to its misincorporation at arginine and tryptophan residues. Our measurements are consistent with the existence of a cluster of ribosomal proteins having an average mistranslation frequency of 2.5 × 10-4/residue and another having an average mistranslation frequency of 10-3 residue. There was three times less cysteine misincorporated into ribosomal protein LI than into L7/L12, although the LI mRNA contains eleven CGU/C codons and four UUU/C codons while the L7/L12 mRNA contains only one arginine and two phenylalanine codons (both proteins are free of tryptophan). Furthermore, the mRNAs for both LI and L7/L12 contain a CGU codon located in the context GUA-codon-GG and there was as much cysteine incorporated at this codon in L7/L12 [Bouadloun, F., Donner, D. and Kurland, C. G. (1983) EMBO J. 2, 1351 -1356] than in the whole of Li. This suggests that, relatively speaking, little cysteine is to be found at the phenylalanine and the other ten arginine positions of LI and that the phenylalanine residues of L7/L12 are particularly error-prone. [ABSTRACT FROM AUTHOR]

Presents data which show that the arrangement of tRNA in the ribosome depends not only on which of the two mRNA codons inside the ribosome binds tRNA but also on the functional and the structural state of the elongating complex. Advantages for the more precise and more detailed classification of the tRNA-binding sites of the ribosome; Interaction with the functional state.

1. An 80S initiation complex was formed by incubating a heterologous cell-free system with 125I-labeled globin mRNAs in the presence of sparsomycin. The 80S initiation complex was then digested with micrococcal nuclease. The ribosomal 5S-RNA · L5-protein (5S RNP) fraction, released by EDTA treatment, contained 125I-labeled mRNA fragments. The attachment of labeled mRNA fragments to 5S RNP was shown by (a) CsCl isopycnic centrifugation, (b) recentrifugation through a sucrose density gradient and (c) acrylamide gel electrophoresis of 5S RNP purified by (b). Labeled fragments were released from 5S RNP by treatment with sodium dodecyl sulfate or pronase, indicating the participation of protein L5 in the attachment. The attached mRNA fragments were 23-25 nucleotides in length. 2. Hybridization experiments, using restriction fragments of cDNA for rabbit β globin mRNA, showed that the attached mRNA fragments were derived from the 5′ portion of globin mRNAs. 3. The attachment of 125I-labeled mRNA fragments to 5S RNP was also observed in the 80S initiation complex formed by incubation of reticulocyte lysate with 125I-labeled globin mRNA, but not in labeled polysomal fractions. These findings may indicate that 5S RNP interacted with the 5′ portion of globin mRNA, containing the translation initiation codon of globin mRNA in the 80S initiation complex. The biochemical significance of these results is discussed. [ABSTRACT FROM AUTHOR]

Reports on the cleavage of rRNA by the treatment of ribosomes in reticulocyte lysates with low concentrations of the calcium-dependent nuclease from Staphylococcus aureus. Localization of the positions of the cleaved phosphodiester bonds by primer extension and polyacrylamide gel electrophoresis; Reason for the decrease in translational activity observed in lysates treated with low amounts of S. aureus nuclease; Suggestion that the induced cleavages were not detrimental to ribosomal function but could influence the rate of ribosomal movement along the messenger RNA.

We have measured the levels of translatable and mRNA and ribosomal protein (r-protein) mRNAs in Tetrahymena pyriformis during nutritional shifts. After 15 min of starvation total mRNA is reduced 2-fold, and after 24 h 7.5-fold, relative to exponentially growing cells. Upon refeeding total mRNA increases rapidly reaching the level of exponentially growing cells after 2.5 h. The levels of the individual r-protein mRNAs are coordinately regulated throughout a starvation-refeeding cycle. The relative levels of r-protein mRNAs remain virtually unchanged during the first hour of starvation and then decrease gradually to 30% of the relative levels in exponentially growing cells. Following refeeding the relative levels of r-protein mRNAs increase 6-fold. Taking into account the changes in whole cell RNA, we have calculated that long-time-starved Tetrahymena cells contain only 4% whereas cells after 3 h of refeeding contain 200% of the amount of r-protein mRNA in exponentially growing cells. The amount of r-protein mRNA thus increases 50-fold during the first 3 h of refeeding. A comparison between the relative levels of r-protein mRNAs and the relative rate of r-protein synthesis in vivo indicates that Tetrahymena employs a combination of control of translation and control of the level of r-protein mRNAs to ensure a rapid reduction in the rate of r-protein synthesis during the early period of starvation. In this period translation of r-protein mRNAs is preferentially inhibited. During refeeding the increase in the rate of r-protein synthesis parallels the increase in the abundance of r-protein mRNAs. [ABSTRACT FROM AUTHOR]

Hepatic microsomal cytochrome P-450 was elevated in 17-day chick embryos by combined administration of the porphyrinogenic drugs 2-allyl-2-isopropylacetamide and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Increased apoprotein levels were the result of de novo protein synthesis; in vitro obtained translation data suggested that cytochrome P-450 mRNA levels were elevated. A 1000-base cDNA sequence for the drug-induced cytochrome P-450 mRNA was isolated from a chick embryo cDNA ‘library’ and this was used as a specific probe to investigate drug-mediated induction of cytochrome P-450 mRNA. RNA-DNA ‘dot’ hybridisation studies demonstrated that drug treatment led to a 3—5-fold increase in the level of this mRNA and that the mRNA was predominantly associated with membrane-bound polyribosomes. Treatment of embryos with the drugs individually demonstrated that both of them induced synthesis of the same mRNA. These studies show directly that treatment of chick embryos with 2-allyl-2-isopropyiacetamide or 3,5- diethoxycarbonyl-1,4-dihydrocollidine caused increased levels of cytochrome P-450 mRNA and suggest that this involved increased transcription of the gene. [ABSTRACT FROM AUTHOR]

A length of 990 ± 80 nucleotides was estimated for the poly(A)-rich mRNA of the higher-molecular-weight precursor of rat liver cytochrome c oxidase subunit IV. Assuming a molecular weight of 19 500 for the cytochrome c oxidase subunit IV precursor, a non-coding region of about 350 nucleotides must exist. In experiments on the site of synthesis it is shown that the poly(A)-rich RNA for the higher-molecular-weight precursor of cytochrome c oxidase subunit IV is found in free, loosely and tightly membrane-bound polyribosomes. This finding does not agree with the present picture of free polysomes as the exclusive site of synthesis for mitochondrial proteins. [ABSTRACT FROM AUTHOR]

Characterizes the proteins associated with duck-globin messenger RNA and its polyadenylated segment in polyribosomal and repressed free messenger ribonucleoprotein complexes. Protein composition in different ionic strength conditions; Protein interaction with non-ribosomal globin mRNA.

Two populations of free messenger ribonucleoprotein (mRNP) particles, sedimenting at 20 S and 40 S respectively, were isolated from a rat liver postpolysomal supernatant. After treatment with 0.5 M KCl and recentrifugation through a sucrose layer, the mRNP particles were characterized with respect to their low-molecular-weight RNA and protein components. 40-S and 20-S particles show very different RNA patterns. Four distinct low-molecular-weight RNA species of approximately 105, 139, 187 and 256 nucleotides were found as components of the 40-S mRNPs. The 20-S mRNP particles contain one major low-Mr RNA species of approximately 243 nucleotides and a characteristic pattern of low-Mr RNAs similar to the one found in nuclear ribonucleoprotein particles. In contrast to the low-Mr RNAs found in nuclear RNP particles most of the low-Mr RNA species present in 20-S and 40-S mRNP particles are rapidly labeled after [3H]orotate administration. Whereas the low-Mr RNA composition of 20-S and 40-S mRNP particles is very different, the protein patterns of both mRNP complexes are very similar. Six major polypeptides with the following molecular weights of 117000, 79800, 76700, 53800, 43900, 36300 and several minor ones were found in both 20-S and 40-S mRNPs. In a cell-free system from wheat germs neither 20-S nor 40-S mRNP particles stimulated the incorporation of [3H]leucine into proteins. However, phenol-extracted RNA from 20-S and 40-S mRNPs stimulated total protein synthesis 16-fold and 3-fold, respectively. Furthermore, the RNA from both mRNP pools directed the synthesis of albumin in vitro. [ABSTRACT FROM AUTHOR]

Crosslinking between phenylalanyl transfer RNA from brewer's yeast and poly(U) can be obtained when ribosomal complexes are irradiated at »300 nm; the ribosomal complexes were formed by enzymatic binding of the tRNA. The crosslinked product was isolated on oligo(dA)-cellulose columns. The experiments suggest that the photo-crosslinking occurs via the wybutine in the anticodon loop of the tRNA. This system can be used to study interactions between tRNA and messenger RNA on the ribosome. [ABSTRACT FROM AUTHOR]

Modification of Escherichia coli ribosomes with phenylglyoxal and butanedione, protein reagents specific for arginyl residues, inactivates polypeptide polymerization, assayed as poly(U)-dependent polyphenylalanine synthesis, and the binding of poly(U). Inactivation is produced by modification of the 30-S subunit. Both the RNA and the protein moieties of 30-S subunits are modified by phenylglyoxal, and modification of either of them is accompanied by inactivation of polypeptide synthesis. Modification of only the split proteins released from 30-S subunits by prolonged dialysis against a low-ionic-strength buffer, which contain mainly protein S1, produces inhibition of poly(U) binding and inactivation of polypeptide synthesis. Amino acid analysis of the modified split proteins showed a significant modifications of arginyl residues. These results indicate that the arginyl residues of a few 30-S proteins might be important in the interaction between mRNA and the 30-S subunit, which agrees with the general role assigned to the arginyl residues of proteins as the positively charged recognition site for anionic ligands. [ABSTRACT FROM AUTHOR]

The native ribosomal subunits of the mouse plasmacytoma cell line MPC-11 were fractionated by CsCl gradient centrifugation into seven classes of particles designated p1, p2, p3, p4, p5, p6 and p7, which possessed densities of approximately 1.38, 1.43, 1.46, 1.49, 1.50, 1.51 and 1.53 g/cm³ respectively. The RNA was analysed by oligo(dT)-cellulose chromatography and gel electrophoresis. In both well-fed and starved cells p3 was generally the dominating class of particles. During metabolic shift-up conditions, however, there was a great increase of p6 particles. Transfer of material containing rRNA and mRNA into this region of the CsCl gradient indicates the accumulation of an initiation complex containing mRNA. During metabolic shift-down conditions a decrease of p6 particles and an increase of p2 and p3 particles was observed. The p2 fraction was rich in mRNA and may contain considerable quantities of messenger ribonucleoprotein particles. A substantial fraction of newly synthesised mRNA-containing material in starved cells banded in fact in the p2 region of the CsCl gradients. Circumstantial evidence indicates the existence of a transient 40-S initiation complex which bands in the p2 — p3 region of the gradients, Double-labelling experiments demonstrated that radioactivity in 40-S subunits first appeared in precursor particles with a density of 1.49 – 1.50 g/cm³ and only later in p3 and p6 particles, p7 particles were detected in starved cells. They were slowly labelled and were presumably derived from old ribosomes following completion of the polysome cycle. [ABSTRACT FROM AUTHOR]

Xenopus laevis oocytes (stage II-III) incorporate [35S]methionine into ribosomal proteins identifiable by two-dimensional electrophoresis. Poly(A)-rich RNA prepared from this source has been translated in a wheat germ cell-free system. Among the products synthesized in vitro 30 are clearly identifiable as ribosomal proteins. Some are not individually resolved, whereas a few of them may lack methionine or may not be correctly processed after the synthesis in vitro. An enrichment of our mRNA preparation for ribosomal protein activity can be obtained by subsequent passages on oligo(dT)-cellulose columns and by selection of a 10-16-S fraction by sucrose gradient sedimentation. We estimate that messenger RNA for ribosomal proteins represents 10-20% of this RNA preparation and can now be utilized for molecular cloning procedures. [ABSTRACT FROM AUTHOR]

In order to identify and to study the ribosomal protein genes in yeast we have tried to purify the mRNA coding for ribosomal proteins. Poly(A)-containing RNA from the yeast Saccharomyces carlsbergensis was fractionated according to size using preparative sucrose gradient centrifugation. The various (size) fractions were translated in vitro in a wheat germ cell-free system. The products were analysed by sodium dodecylsulfate/polyacrylamide gradient gel electrophoresis as well as by acetic acid/urea gel electrophoresis. It was found that an mRNA fraction of about 9 S directs the synthesis in vitro of proteins that have properties characteristic of ribosomal proteins, i.e. they are both small and basic. The ribosomal nature of these proteins was further established by two-dimensional gel electrophoresis. This small-size mRNA fraction can be used as a probe for the identification of ribosomal protein genes in recombinant DNA molecules. [ABSTRACT FROM AUTHOR]

Substantial evidence is provided to corroborate our previous finding that Escherichia coli ribosomes recognize two binding sites on the 5′ end of alfalfa mosaic virus (AMV) RNA 4 [for a preliminary report see Castel, A., Kraal, B., Kerklaan, P. R. M., Klok, J., and Bosch, L. (1977) Proc. Natl Acad. Sci. U.S,A. 74, 5509–5513]. Translation can start at either site using AcPhe-tRNA or fMet-tRNA as initiator and takes place in the same reading frame along the monocistronic mRNA. The size and composition of the isolated extra NH2-terminal fragment of the acetylphenylalanyl product were found to be in agreement with the 5′ non-coding region of the messenger. Removal of the 5′-terminal cap structure of AMV RNA 4 did not influence significantly both initiation reactions. Ribosomal protein S1 was essential for binding as well as incorporation of both fMet-tRNA and AcPhe-tRNA. A similar interaction on the ribosome was found for AcPhe-tRNA directed by AMV RNA 4 as for fMet-tRNA directed by either AMV RNA 4 or MS2 RNA with respect to the influence of initiation factors. It is concluded that the heterologous plant viral messenger is reliably translated in the E. coli system and that E, coli ribosomes recognize with high specificity an extra initiation site close to the 5′ extremity of the messenger. The relation- ship of this site to a hypothetical entry site involved in the early recognition in the initiation mechanism between ribosome and messenger is discussed. [ABSTRACT FROM AUTHOR]

Additional of phytohaemagglutinin to cultured lymphocytes results in a progressive increase in the rate of protein synthesis of up to 7–10 times the initial rate after 20 h. Between 2 h and 12 h after the addition of mitogen, the increase in the rate of protein synthesis can be accounted for by the transfer of mRNA from messenger ribonucleoprotein complexes to polysomes. Between 12 h and 20 h the increase also reflects the accumulation of mRNA and ribosomes. However, the proportion of mRNA associated with ribosomes in unstimulated lymphocytes is unexpectedly high and paradoxically decreases during the first 2 h after mitogen addition, although the rate of protein synthesis increases. A mechanism involving mRNA selection is suggested. [ABSTRACT FROM AUTHOR]

Normal human lymphocytes stimulated by phytohemagglutinin P, and other mammalian cells were rendered permeable to macromolecules such as poly(U) and proteins, by treatment with a low concentration of toluene. Under this condition, poly(U) translation was more efficient in the permeabilized cells than in 10000 × g extracts. Such a process occurs inside the treated cells as demonstrated by the fact that [14C]uridine-labelled ribosomes remain associated with the toluene- treated lymphocytes even after incubation at 37 °C. A nuclease from Staphylococcus aureus was able to penetrate the permeabilized cells and to break the polysome-bound endogenous messenger RNA. However, the protein-synthesizing machinery inside the toluene-treated lymphocytes was unaffected by the nuclease, as demonstrated by the unimpairement of polyphenylalanine synthesis when poly(U) was added after the preincubation with the enzyme. These results suggest that the toluene treatment can be considered as an important tool for the study of the synthesis of macromolecules and its regulation in eukaryotic cells. [ABSTRACT FROM AUTHOR]

The effects of ultraviolet irradiation on the rates of synthesis of individual ribosomal proteins in yeast were examined and compared with the ultraviolet sensitivities of the synthesis of other yeast proteins. It was found that the synthesis of yeast ribosomal proteins is much more sensitive to ultraviolet irradiation than that of other yeast cellular proteins. Taking into account the half-life of yeast mRNA, the results obtained indicate that the genes coding for ribosomal proteins form part of long transcriptional units, which are much longer than the DNA stretch needed to code for a ribosomal protein of average molecular weight. Saturation hybridization of total poly(A)-containing mRNA with yeast nuclear DNA revealed that as much as 30% of DNA is complementary to yeast mRNA. Thus, the primary transcript of a protein gene on the average is about 1.7 times the length of the actual messenger. On the basis of the various experimental data we suggest a clustering of the yeast ribosomal protein genes in a number of common transcriptional units. [ABSTRACT FROM AUTHOR]

Poly(4-thiouridylic acid) [poly(s4U)] synthesized by polymerization of 4-thiouridine 5′-diphosphate with Escherichia coli polynucleotide phosphorylase (EC 2.7.7.8) acts as messenger RNA in vitro in a protein-synthesizing system from E. coli. It stimulates binding of Phe-tRNA to ribosomes both in the presence of EF-Tu · Ts at 5 mM Mg2+ concentration and nonenzymatically at 20 mM Mg2+ concentration. It codes for the synthesis of polyphenylalanine. Poly(s4U) competes with poly(U) for binding to E. coli ribosomes. Light of 330 nm photoactivates poly(s4U) thus making it a useful photoaffinity label for the ribosomal mRNA binding site. Upon irradiation of 70-S ribosomal complexes, photoreaction occurs with ribosomal proteins as well as 16-S RNA. Ribosomes preincubated with R17 RNA are protected against the photoaffinity reaction. The labelling of 16-S RNA can be reduced by treatment of ribosomes with colicin E3. [ABSTRACT FROM AUTHOR]

There is a single-site interaction of [methylene-14C]thiamphenicol and [methylene-14C]chlor-amphenicol with run-off ribosomes with dissociation constants Kd = 6.8 μM and Kd = 4.6 μM respectively. Similar affinities for the antibiotics are observed in polysomes totally deprived of nascent peptides, or bearing nascent peptides on the A-site. However, two types of interaction are observed in endogenous polysomes with some ribosomes bearing nascent peptides on the P-site and others in the A-site. The lower-affinity bindings (dissociation constants Kd = 6.4 μM and Kd = 5.9 μM for thiamphenicol and chloramphenicol respectively) are due to the ribosomes bearing nascent peptides on the A-site. The higher-affinity bindings (dissociation constants Kd = 2.3 μM and Kd = 1.5 μM for thiamphenicol and chloramphenicol, respectively) are due to the ribosomes bearing nascent peptides on the P-site. Therefore binding of nascent peptides to the A-site does not affect the affinities of thiamphenicol and chloramphenicol for the ribosome. On the other hand interaction of the nascent peptides with the P-site of the ribosomes increases the affinities of both antibiotics for the ribosome. Thiamphenicol and chloramphenicol are thus good inhibitors of peptidebond formation in ribosomes and polysomes. Their affinities are increased precisely when the peptidyl-tRNA is placed in the P-site preceeding the peptide bond formation step, which is specifically blocked by the antibiotics. There is a single-site interaction per ribosome for [35S]thiostrepton, which does not appear to be affected by the attachment to the ribosomes of mRNA, tRNA and nascent peptides either to the A or the P-site. [N-methyl-14C]Lincomycin, [N-methyl-14C]erythromycin, [G-3H]streptogramin B and [G-3H]- streptogramin A bind to run-off ribosomes and polysomes totally free from nascent peptides. However, these antibiotics do not interact with ribosomes bearing nascent peptides either in the A or the P-site and therefore are not active on preformed polysomes. Thus lincomycin and streptogramin A only interact with free ribosomes and 50-S subunits and block the early rounds of peptide bond formation prior to polysome formation. Erythromycin and streptogramin B do not inhibit either initiation or the first round of peptide bond formation. However, erythromycin and streptogramin B, prebound to the ribosome, block peptide elongation probably by steric hindrance with the growing oligopeptide chain when this reaches a certain critical length. [ABSTRACT FROM AUTHOR]

In order to elucidate the mechanism of action of inhibitors that block the initiation of protein synthesis in mammalian systems, we have studied the following steps: (a) formation of the ternary complex Met-tRNAf · IF-E2 · GTP, (b) binding of the initiator Met-tRNAf to the 40-S ribosomal subunit in the presence of initiation factors and dependent or not on the addition of mRNA, (c) formation of the initiation complex with 80-S ribosomes and (d) formation of the first peptide bond. Adrenochrome, aurintricarboxylic acid, polydextran sulphate, pyrochatechol violet and show-domycin block the formation of the ternary complex Met-tRNAf · IF-E2 · GTP. Edeine A1, aurintricarboxytic acid and polydextran sulphate block the binding of the mRNA to the 40-S ribosomal subunit. Pactamycin induces the formation of stable smaller initiation complexes which are unable to go through the subsequent steps of initiation. Stimulation of the binding of the initiator Met-tRNAf to the 80-S ribosome in the presence of initiation factors is observed with sparsomycin and antibiotics of the sesquiterpene family (verrucarin A, trichodermin and trichothecin). However, these antibiotics block the reaction of the bound Met-tRNAf with puromycin. Narciclasine has no effect on the binding of the initiator to the ribosome but strongly blocks its reaction with puromycin. We have developed a simple technique to detect the Met-tRNAf · 40-S-subunit · poly(A,G,U) initiation complexes by chromatography on Sepharose 6B columns. The requirements for the formation of such complexes measured by this technique and its comparison with the sucrose gradient centrifugation method are described. [ABSTRACT FROM AUTHOR]

16-S ribosomal RNA and a complex of ribosomal protein S4 and 16-S rRNA were studied in solution by small-angle X-ray scattering. Concentration series of the 16-S rRNA and the S4 · 16-S-rRNA complex were measured in 37.5 mM Tris-HCl buffer pH 7.4 at 5°C. The following data were determined. The radii of gyration for the 16-S rRNA and S4 · 16S-rRNA complex were R = 17.6 ± 0.4 nm and 17.5 ± 0.6 nm, respectively. The two respective values of the radii of gyration of the cross-section were Rq.1 = 8.42 ± 0.1 nm and 8.33 ± 0.3 nm, and Rq.2 = 0.988 ± 0.03 nm and 0.996 ± 0.03 nm. The largest diameters of the 16-S RNA and S4 · 16-S-RNA complex were L = 61.8 ± 1 nm and 60.0 ± 1 nm, respectively. Volumes of V = 1570 ± 60 nm³ were found for both particles. In the Tris buffer used, no significant differences were found between the scattering curves of 16-S rRNA and the S4 · 16-S-rRNA complex. A model equivalent in scattering properties to the RNA and complex is a flat elliptical cylinder with the following dimensions: large axis 61.7 nm, small axis 35.4 nm and height 2 nm. The theoretical scattering curve fits the experimental one as long as the shape of the measured curve is due to the overall shape of the particle. A model equivalent in scattering over the whole measured angular range is one built up from a large numbe of spheres that simulate the known substructure of the RNA. The outer dimensions of this model correspond to those of the flat elliptical cylinder. [ABSTRACT FROM AUTHOR]

With the use of ³H-labeled R17 amB2 phage RNA having an UAG codon at the seventh triplet of the coat cistron, release of the RNA from ribosomes at the termination codon was studied. The ribosome-releasing factor previously described was shown to stimulate the process of mRNA release at the termination codon. The stimulatory effect of the factor on this process was dependent on the termination factor (RF-1). GTP was required for this process and guanosine 5'-(β,γ-methylene)triphosphate could not replace GTP. No apparent change of size of R17 RNA was observed during the release of the R17 RNA from the ribosomes. The ribosome-releasing factor is distinct from the known termination codon-specific factor such as RF-1. [ABSTRACT FROM AUTHOR]

When rabbit reticulocyte polyribosomes are treated with 0.5 M KCl, they dissociate into subunits and release a protein fraction which is required for peptide chain initiation in a cell-free system using KCl­treated subunits as the source of ribosomes. Three independent methods were used to determine the fate of mRNA after KCl treatment of the subunits. These three methods (sucrose gradient analysis of RNA after dissociating it from protein with sodium dodecylsulfate, acrylamide gel electrophoresis of RNA and electron microscopic analysis of subunits) all showed the 8–9-S mRNA to be associated with the small subunit, but not the large subunit. Furthermore, no mRNA was found to be associated with either ‘native’ ribosomal subunit in a reticulocyte lysate. [ABSTRACT FROM AUTHOR]

The nature of the interaction of membrane-bound polysomes with the endoplasmic reticulum has been studied in MOPC 21 mouse plasmocytoma tissue-culture cells which secrete an immunoglobulin G. Treatment of microsomes at high salt concentrations (500 mM KCl) results in the detachment of 40-45 % of the bound ribosomes. This released ("loose") fraction is predominantly monosomal in contrast to the remaining "tight" polysomes. It is shown that both ribosomal fractions contain immunoglobulin light-chain mRNA which argues against a functional distinction between them in the cell. Polysomes have been dissociated to apparently undegraded ribosomal subunits at 0 °C by incubation with puromycin in the presence of a magnesium ion buffer. Application of this procedure to membrane-bound polysomes results in partial release of ribosomal subunits from the membrane. The ribosomal material which is not so released sediments predominantly at 60 S and this fraction can be detached by subsequent washing at 500 mM KCl. Thus, by use of the polysome-dissociating procedure together with high salt concentrations, essentially total release of the ribosomal components can be obtained. The results indicate that the salt-labile interactions are mediated via the large ribosomal subunits and are the only anchoring forces to the membrane at early stages of protein synthesis. The tight membrane-bound polysomes result from the additional anchoring effect of the polypeptide chains as they grow through the membrane. [ABSTRACT FROM AUTHOR]

The attachment of derived ³H-labelled rat-liver 40-S subparticles to haemoglobin mRNA, present in the polysomes of a rabbit-reticulocyte lysate, has been studied by sucrose-gradient analysis. The attachment is dependent on time, energy and temperature, is reduced by cycloheximide and abolished by aurintricarboxylic acid. The resulting "hybrid" polysomes appear as active as the endogenous polysomes in protein synthesis. It is concluded that in an unfractionated-reticulocyte lysate, heterologous 40-S subparticles may become incorporated into polysomes without any additional factors. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) and messenger like RSNA (mlRNA) were investigated in the cytoplasm of HeLa cells while ribosomal RNA synthesis was arrested. Under these conditions, functional mRNA associated in polyribosomes and cytoplasmic free mlRNA are formed and can be labelled selectively to steady state. All cytoplasmic non-ribosomal RNA sedimenting at more than 6-7 S exists in the form of ribonucleoprotein complexes which pre-exist in the cell, and are stable upon cell lysis, sedimentation and (after fixation) CsC1 density gradient analysis. The functional, true mRNA is contained in a complex of mRNA and protein which bands in association with ribosomes (... = 1.52 to 1.60 g/cm³) in CsC1 density gradients or, released by EDTA, at its own intrinsic density of 1.40-1.48 g/cm³. The cytoplasmic free mlRNA bands as a particle of mlRNA-containing ribonucleoprotein at an identical low density. The molecular weight spectrum of mRNA is identical to that of mlRNA and the sedimentation pattern of the mRNA, protein complex released from polyribosomes is similar to that of the free mlRNA, protein complex. The physico-chemical separation of mRNA · and mlRNA · protein complexes allowed us to follow their relative kinetics of synthesis and decay. Each type of ribonucleoprotein obeys a different, strictly time-dependent pattern. Label enters the pool of free mlRNA · protein complexes first and may, in a pulse-chase experiment, be partially chased into polyribosomes. At steady state (6 h) 40-60% of the labelled RNA remains in the form of free mlRNA · protein particles. These cannot be chased into polyribosomes, the kinetics of mRNA · and mlRNA · protein complexes decay following identical patterns. These firings are in agreement with a model according to which mlRNA from the nucleus first joins the pool of free ribonucleoprotein. Then, the activated mRNA · protein complexes attach to ribosomal subunits and form polyribosomes whereas inactivated mlRNA · protein complexes remain free in the cytoplasm. In order to further strengthen the evidence in favour of the real existence of mRNA · and mlRNA · protein complexes in the cells, the corresponding fractions from sedimentation or CsCI density gradients were observed in the electron microscope. By this method it was possible to see small cytoplasmic particles which have not before been identified. The rounded structures, with diameters ranging from 100 Å to 200 Å, seem to consist of the coiling of a 35 Å wide pearllike chain which may also be identified in polyribosomes. The frequency of these particles is highest in the mlRNA · protein band (... = 1.40-1.48 g/cm³). Thus they may correspond to the mRNA. protein complex. However since they share some morphological features with other known biological structures the evidence is not conclusive. [ABSTRACT FROM AUTHOR]

Polyribosomes containing mRNA differentially labelled with [³H]uracil and [14C]uracil in the 5′-and the 3′-proximal portions were isolated from Escherichia coli cells, mRNA degradation to alcohol-soluble products occurred when polyribosomes were incubated in conditions of protein synthesis and proceeded in the 5′ to 3′-direction. 15% of the mRNA was degraded at the end of a 10-min period of incubation. The mRNA-degrading enzyme (or enzymic system) was found to be bound to polyribosomes; it could be removed by washing in low salt. The content in RNAase activity per ribosomes in polyribosomes increased three to five-fold when polyribosomes were isolated from cells in which extensive run-off of ribosomes from mRNA had been induced. On the contrary the RNAase activity in free ribosomes was not affected. This is taken as an indication that the enzyme(s) responsible for the degradation of endogenous mRNA in vitro is not aspecifically adsorbed to ribosomes, but specifically located towards the 5′-end of the mRNA chains in polyribosomes. [ABSTRACT FROM AUTHOR]

Two RNA · protein complexes were isolated from duck erythroblast postribosomal supernatants. Their nominal sedimentation values on sucrose gradients are 12 S and 20 S, respectively. The 12-S particle contains a 4–6 S RNA, in the 20-S particle a 9-S RNA is found. This 9-S RNA is shown to direct the synthesis of all duck globin chains in a cell-free, messenger RNA-dependent protein-synthesizing system. The protein moiety of these ribosome-free particles is described and compared with the proteins found in the mRNA · protein complex liberated by EDTA from polyribosomes. We show that in the free-cytoplasmic particles no protein can be found which is identical to any of the polypeptides associated with polyribosomal mRNA. Some of these proteins are phosphorylated and contain phosphoserine. The electrophoretic patterns of phosphorylated proteins from the two globin mRNA-containing complexes differ, as do those of the unlabelled polypeptides. We conclude that the mRNA-associated protein population is exchanged when the mRNA enters the translation machinery. The possible role of the RNA-associated proteins in the post-transcriptional and translational control of eukaryotic protein synthesis is discussed. [ABSTRACT FROM AUTHOR]

Messenger ribonucleic acid was isolated from calf lens polyribosomes and fractionated. Previous identifications of the newly synthesized products have been extended in that the fidelity of the translation of the 14-S mRNA was assayed by fingerprinting analysis. The results suggest that lens messenger is translated with a high degree of fidelity in the cross systems even to the extent that the newly synthesized αA2 chains contain an acetylated methionine residue in the N-terminal position like the native chains. There seems to be no requirement for cell-specific protein factors in order to obtain accurate translation. Evidence is accumulating that the acetylation is not involved in the initiation mechanism. [ABSTRACT FROM AUTHOR]

The effect of the purified initiation factors F1, F2 and F3 on the binding of radioactive dihydrostreptomycin to 30-S subunits and 70-S sensitive ribosomes of Escherichia coli was studies in the absence of other substrates. Neither of the three factors alone inhibits dihydrostreptomycin binding on 30-S subunits: the same applies to 70-S ribosome except that F2 alone exerts a partial inhibitory effect when present in large excess. In contrast certain associations of factors appear very efficient in prevention dihydrostreptomycin from binding on ribosomes: thus a mixture of F1 with either F2 or F3 strongly inhibits radioactive dihydrostreptomycin binding to 70-S ribosomes: the same is observable with 30-S subunits although the F1-F3 association exerts the most potent effect, this effect being even more pronounced by the further addition of F2. The competing effects exerted by factors toward streptomycin binding does no manifest itself at low temperatures. Elongation factors have no effect. In line with these results, it was also found that combination of the three initiation factors can protect ribosomes against the streptomycin-induced release of initiator tRNA which is bound in the presence of natural messenger-RNA and GTP. The observed competition between initiation factors and streptomycin for the ribosomes indicates that the factors either blind or induce conformational changes in th P10 region of the ribosome. [ABSTRACT FROM AUTHOR]