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Peptide studies on two acidic proteins, A1 and A2, from 50-S ribosomes of Escherichia coli indicate a closely related primary structure. 1. Amino acid compositions of the tryptic peptides of the two proteins are identical, with exception of the amino terminal peptide. 2. The N-terminal amino acid sequence of A2-protein is Ser-Ile-Thr-Lys, while that of A1-protein is N-acetyl-Ser-Ile-Thr-Lys. 3. The estimated number of 120 amino acid residues in each polypeptide chain is consistent with the physical molecular weight estimate. 4. In 50% of the polypeptide chains, in both A1- or A2-protein, a specific lysine residue is replaced by ε-N-monomethyl-lysine. [ABSTRACT FROM AUTHOR]

Two isogenic derivatives of Escherichia coli K12: strains EA 1 (Met-, Biot-, RCstr and EA 2 (Mei-, Biot-, RCrel) when grown in the presence of ethionine in place of methionine, synthesize ribosomes whose RNA is submethylated and whose proteins contain ethionine. Net ribosome synthesis in the presence of ethionine is greater in the RCrel strain than in the RCstr strain. The precedinig paper describes results obtained with the latter strain. Here we present further studies carried out with the isogenic RCrel strain EA 2 As in the case of the RCstr strain (previous paper) ribosomes synthesized by the RCrel strain grown in the presence of ethionine display defective association properties. A large fraction of these "ethionine" ribosomes is found as free subunits in crude extracts containing 10 mM Mg+ prepared from strain EA 2 grown in an ethionine-containing medium. These free "ethionine" subunits have sedimentation coefficients of 28 S and 45 S approximately while the "ethionine" subunits found associated as 70 S particles in crude bacterial extracts sediment at 30 S and 50 S respectively. Both classes of "ethionine" subunits (free, and associated) can be methylated in vitro in the presence of S-adenosyl methionine with or without the addition of an external source of methylases (100000 x g supernatant of a homologous crude bacterial extract) The latter observation shows that the "ethionine" subunits contain bound methylases. These enzymes do not seem to be normal ribosomal proteins nor to be bound specifically to "ethionine" ribosomes since (a) they are removed by washing the "ethionine" ribosomes with 1 M NH4Cl, and (b) they are found bound to normal methionine ribosomes from which they are also removed by washing with 1 M NH4Cl. [ABSTRACT FROM AUTHOR]

The two types of 30-S · 50-S couples described in the preceding paper, differ markedly in their sensitivity to hydrostatic pressure evoked during centrifugation in sucrose gradients. The sedimentation coefficient of about 60 S recorded with couples formed from native subunits is only apparent, the real a value being 70 S. The latter couples can be stabilized against pressure-induced dissociation by bound tRNA. On the other hand this binding renders couples formed from derived subunits more sensitive to this dissociation. As the factor determining this intriguing difference in pressure sensitivity lies in the 50-S subunit, a comparative analysis of a number of ribosomal constituents has been performed for native and derived 50-S particles. No qualitative differences in the proteins L1–L34 have been detected. Quantitative differences cannot yet be excluded. It is considered unlikely that differences in rRNAs or low-molecular-weight components arc responsible for the distinct association behaviour. Partial reconstitution of ribosomal γ-cores and split proteins detached by CsCl reveals that the determinant factor is located in the core particles. This suggests that native and derived 50-S subunits do not differ in conformation only. The possibility may be envisaged therefore, that the two types of 50-S differ in a ribosomal protein. [ABSTRACT FROM AUTHOR]

The binding of purified Gly3-Phe-tRNA to Escherichia coli ribosomes was compared to that of crude Gly3-Phe-tRNA (containing bulk tRNA). Gly3-phe-tRNA from the purified preparation binds preferentially to the P site (peptidyl site) at both a high (30 mM) and a low (10 mM) magnesium acetate concentration, while peptidyl-tRNA from the crude preparation bound more to A site (aminoacyl site) than to the P site at 30 mM magnesium acetate under the same experimental conditions. The addition of tRNAPhe to the purified Gly3-Phe-tRNA preparation strongly inhibited the binding at 10 mM. At 30 mM Mg2+ the addition of the same amount of tRNAPhe caused the Gly3-Phe-tRNA to bind partially to the A site, but did not inhibit the total amount of Gly3-Phe-tRNA which binds to the ribosomes. The binding of purified Phe-tRNA in the presence of T factor and GTP is not inhibited by tetracycline. However, tetracycline strongly inhibited the enzymatic binding of Phe-tRNA when tRNAPhe was added. From the results presented in this paper it is proposed that in the presence of poly(U), Phe-tRNA or peptidyl-tRNAPhe can bind to A sites only of those ribosomes which have their P site occupied by tRNA or one of its derivatives (aminoacyl-tRNA or peptidyl-tRNA). [ABSTRACT FROM AUTHOR]

It is well know that protein synthesis stopped either by chloromycetin or by starvation of an essential amino acid for a RC relstrain, ribosomal RNA synthesis continues, but products do not appear in 50 S and 30 ribosomes. It has also been shown that 5 S RNA synthesis is altered by the addition of chloromycetin but in an unknown manner. In this paper we report that: (a) during amino-acid starvation of a starvation of RCrel strain of Escherichia coli, 5 S RNA is transcribed as 5 S relax RNA located outside the ribosomes, in the supernatant fraction; (b) this 5 S RNA which is slightly longer than the normal product is convertible into a true 5 S RNA when protein synthesis is restored after blockage of RNA synthesis by actinomycin ;(c) part of the 23 S RNA from a 25 S particle appears after resumption of protein synthesis in a 40 S parcel which contains normal 5S RNA; (d) a pool of 5 S precursor RNA exists outside the 50 S ribosome. This 5 S precursor RNA is isolated; (e) this precursor which is isolated from the 105000xg supernatant fraction after pulse labeling, migrates on gel electrophoresis between 5 S relax RNA and true 5 S RNA. [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]

The primary structures of the 16S and 23S ribosomal RNAs of Escherichia coli have been studied. Nucleotide sequences have been determined which occur in specific areas close to the small numbers of methylated nucleotides which are present in these molecules. Fragments containing these methylated nucleotides were obtained by digestion of radioactive RNA substrates with T1 ribonuclease. Such digestion products were fractionated by high-voltage electrophoresis in two dimensions. The 23S RNA yielded predominantly 11 methylated oligonucleotides on digestion, and the 16S RNA gave rise to 6 methylated components. Nucleotide sequence analysis of these products was carried out, and the nature of the modified nucleotides occurring in each product was also examined. It was found that some of the sequences contained several modified components positioned close to each other. Quantitative studies indicate that each of the methylated sequences is probably present twice in a molecule of the 23S RNA, so that these sequences encompass about 5% of the nucleotides present in the entire molecule. It is postulated that extensive repetition of nucleotide sequences occurs within the 23S RNA. [ABSTRACT FROM AUTHOR]

The non-enzymatic binding of aminoacyl-tRNA, N-blocked aminoacyl-tRNA, peptidyl-tRNA and N-blocked peptidyl-tRNA to E. coli ribosomes was measured at 0.01 M magnesium ion concentration in the presence of poly U. It was found that, the maximal amounts of phenylalanyl-tRNA and peptidyl-tRNA, that can be bound to the ribosomes are the same, but the amounts of N-blocked phenylalanyl-tRNA and N-blocked peptidyl-tRNA, that can maximally be bound to the ribosomes are much less. Phenytalanyl-tRNA and peptidyl-tRNA can be bound to ribosomes which are saturated in respect to N-blocked phenylalanyl-tRNA or N-blocked peptidyl-tRNA; however, the presence of the N-blocked substrates on the ribosomes partially inhibits the additional binding of phenylalanyl-tRNA or peptidyl-tRNA. Aminoacyl-tRNA added to ribosomes saturated with respect to peptidyl-tRNA, partially displaces the peptidyl-tRNA from its ribosomal binding site. Under the same conditions, peptidyl-tRNA exchanges with prebound aminoacyl-tRNA considerably less. [ABSTRACT FROM AUTHOR]

Effect of spermine on the binding of erythromycin to ribosomes from Escherichia coli (Q13) and on N-acetylphenylalanyl-puromycin (AcPhe-puromycin) synthesis on the ribosomes was studied under various ionic conditions. 1. Spermine inhibited the binding of erythromycin to ribosomes. The inhibitory effect was diminished with increasing concentration of Mg2+ or monovalent cation such as K+ or NH4+. 2. The preincubation of ribosomes with spermine strongly decreased the affinity of ribosomes to erythromycin. The affinity of spermine-treated ribosomes was restored by subsequent incubation with high concentration of NH4+. These inactivation and reactivation processes were strongly dependent on the incubation temperature. 3. At a high concentration of Mg2+ (16 mM), AcPhe-puromycin synthesis was inhibited by the addition of spermine, although the binding of N-acetylphenylalanyl-tRNA (AcPhe-tRNA) to ribosomes was not affected. 4. At a low Mg2+ (5 mM) concentration, AcPhe-tRNA-ribosome complex formation and AcPhe-puromycin synthesis were stimulated by the addition of relatively low concentration of spermine. Further additions of spermine resulted in marked reductions of AcPhe-puromycin synthesis, while AcPhe-tRNA · ribosome complex formation was little affected. 5. The inhibition by spermine of AcPhe-puromycin synthesis was reduced by increasing concentration of K+ or NH4+, regardless of the concentration of Mg2+. These results suggest that spermine binds to ribosomes and the incubation of the spermine · ribosome complex at physiological temperature induces an alteration of ribosome conformation and consequent changes in the ribosomal functions. [ABSTRACT FROM AUTHOR]

16-S RNA and five pure ribosomal proteins (S3, S4, S6, S7 and S8) from Escherichia coli 30-S subunits were studied by circular dichroism in regard to the reconstitution of biologically active subunits. A small structural variation upon heating could be measured for RNA. It cannot be interpreted in terms of stacking or disruption of hydrogen bonds. However, most proteins underwent important structural modifications when heated to 42 °C in reconstitution solvent. This effect was not dependent on the presence of magnesium salt. The relative amounts of secondary structure of native and heat-denatured ribosomal proteins were calculated by using three reference curves obtained from standard proteins, the exact secondary structure of which has been determined by X-ray crystallography. In contrast with earlier results, about 18 %, β-structure was detected in native proteins. This discrepancy can most probably be attributed to the calculation method used. Results are discussed in relation to reconstitution of ribosomes. [ABSTRACT FROM AUTHOR]

50-S ribosomal particles from Escherichia coli were hydrolysed with ribonuclease T1, and the ribonucleoprotein fragments produced were fractionated on polyacrylamide gel. The proteins contained in these fragments were analysed on gels containing the detergent sarkosyl (N-laurylsarcosine), using the technique already published. Two-dimensional gel electrophoresis was used for the final identification of the proteins. Two very specific fragments were obtained, the smaller containing only proteins L1 and L9. and the larger containing only L5, L18 and L25. The latter proteins have been shown by other authors to be involved in binding to 5-S RNA. but in this case the fragment contained more RNA than can be accounted for by 5-S RNA alone. This offers the possibility of determining the regions of 23-S RNA associated both with these two groups of proteins and with 5-S RNA. Both fragments are discussed in terms of a possible oligonucleotide analysis of their RNA moieties. [ABSTRACT FROM AUTHOR]

Application of the two-dimensional polyacrylamide gel electrophoretic procedure recently described by Mets and Bogorad [4] to the resolution of Escherichia coli ribosomal proteins is described. This procedure is fast (running time of 8-10 h) and highly sensitive (1-2 µg protein per spot) and it gave good resolution for the majority of the 55 proteins in E. coli ribosomes. Moreover, the method has permitted the resolution of a large number of high-molecular-weight. proteins associated with ribosomes not exposed to high-salt washing. Using purified proteins, the positions in the electrophrogram of the individual proteins of the 30-S subunit have been determined. [ABSTRACT FROM AUTHOR]

In Escherichia coli cells infected with the bacteriophage MS2, pronounced inhibition of ribosomal RNA synthesis occurs amounting to 50% of that of the uninfected control cells at 30 min post-infection. Over the same period the synthesis of tRNA, mRNA and total protein remains close to the level of uninfected E. coli, yet the synthesis of host protein is gradually replaced by that of viral origin. After 30-35 min post-infection the macromolecular synthesis of all the types under study significantly decreases. It is suggested that suppression of host protein synthesis is caused not by selective inhibition at transcription or translation level, but is rather a result of competition between E. coil mRNA and MS2 RNA. On the other hand, exclusive suppression of only ribosomal RNA synthesis may arise from changes in the regulation of transcription of ribosomal RNA locus.

Phosphatidylserine synthetase occurs in a soluble and a ribosome-bound form in Escherichia coli. The soluble enzyme of E. coli B was partially purified by DEAE-Sephadex chromatography. The ribosomal enzyme was obtained by differential centrifugation. Two types of the enzyme had the same Km (150 μ M) for L-serine. The soluble enzyme was required for the biosynthesis of phosphatidylethanolamine from phosphatidic acid via (CDP-diglyceride in the membrane. The ribosomal enzyme was less effective to the phosphatidylethanolamine formation than the soluble enzyme. By the sucrose-density-gradient centrifugation method the soluble enzyme was proved to be loosely associated with the membrane. [ABSTRACT FROM AUTHOR]

1. The bindings of dihydrostreptomycin and N-acetylphenylalanyl-tRNA to Escherichia coli (Q13) ribosomes, which are dependent upon the concentration of Mg2+, were enhanced by spermine, spermidine or putrescine at low concentrations of Mg2+. However, critical levels of Mg2+ which are specific for each polyamine were essential for the enhancement. 2. When the amount of ribosomes was increased, increasing concentration of spermine was required for the same level of stimulation. 3. The binding of spermine to the ribosomes proceeded rapidly and the amount of spermine bound was independent of the incubation temperature from 0 °C to 37 °C. The stimulation of dihydrostreptomycin binding ability of ribosomes by spermine, however, was dependent upon the temperature and the period of the incubation. 4. When ribosomes were preincubated at 37 °C with spermine and adsorbed on a Millipore filter, the spermine · ribosome complex on the filter could efficiently bind dihydrostreptomycin. Although washing with an ice-cold buffer solution containing a high concentration of NH4Cl removed most of the bound spermine from the spermine · ribosome complex, the ribosomes remained active in binding dihydrostreptomycin. [ABSTRACT FROM AUTHOR]

Association between ribosomal subunits of Eschrichia coli can lead to the formation of two classes of 30-S · 50-S couples. The first class sedimenting between 50 S and 70 S, arises when native subunits associate, the second sedimenting at 70 S upon coupling of so-called derived subunits. From cross experiments it is concluded that it is the 50-S subunit which determines the nature of the association product, whereas the 30-S subunit determines whether an association product can be formed. Native and derived 50-S subunits are indistinguishable by sucrose gradient centrifugation and do not differ qualitatively in their ability to form polypeptides under the directions of bacteriophage RNA. Native 30-8 subunits lose a factor during isolation, probably IF-3. They are therefore able to associate both at 0° and 37 °C. On the other hand derived 30-S subunits are unable to associate at 0 °C but they do so at 37 °C in the presence of 10 mM Mg2+. A number of trivial factors have been excluded to explain the difference between the two types of 50-S subunits. The preparation method is not responsible for the difference. Nor do the derived subunits contain peptidyl-tRNA, known to influence subunit association. The content of tRNA and aminoacyl-tRNA has been determined quantitatively by polyacrylamide-gel electrophoresis. None of the subunits nor the 70-S ribosomes from which the derived subunits are prepared contain a tRNA species. [ABSTRACT FROM AUTHOR]

A method is presented to isolate in pure form two acidic proteins, indicated as A1- and A2-protein from 50-S ribosomes of Escherichia coli. The advantage of this method is that it does not require prior isolation of subunits. The molecular weight of A1- and A2-protein was found by dodecylsulfate gel electrophoresis to be 11 500 with a maxum uncertainty of 1500. Sedimentation equilibrium centrifugation in 5 M guanidine hydrochloride indicates a molecular weight in the range of 12 000-13 000. The amino acid composition of the two proteins is remarkable and indistinguishable. A-protein has a high content of alanine and does not contain tyrosine, tryptophan, histidine or cysteine. On gel-electrofocusing the isoelectric points of A1- and A2-protein are slightly different, namely pH 4.7 and pH 4.85. The amount of A-protein in actively diving cells indicates that contrary to 30-S ribosomes, 50-S ribosomes have a restricted element of repeat. The number of copies of A-protein is found to be somewhat larger than two. [ABSTRACT FROM AUTHOR]

50 S ribosomal subunits of Escherichia coli obtained by zonal centrifugation in B XV rotors were treated with various concentrations of LiCl in the absence and presence of urea. This treatment resulted in three protein fractions, each of which was subjected to CM-cellulose chromatography. Gel filtration in Sephadex G-100 (and preparative polyacrylamide electrophoresis) were used for further separation. The purity of the isolated proteins was better than 95% as shown by four methods. Proteins were isolated in yields of 6-90 mg depending mainly on the number and kind of purification steps. Instead of fractionation with LiCl prior to column chromatography, precipitation of extracted proteins at various concentrations of ammonium sulfate was tested. This method is useful if a given protein, e.g. from mutants is wanted in large quantity. [ABSTRACT FROM AUTHOR]

In Escherichia coli there is one binding site per ribosome for lincomycin, chloramphenicol and erythromycin. These antibiotics act at closely related sites on the 50-S subunit. There are, however, some interesting differences m the properties of the three sites, such as: (a) the apparent possibility of obstructing the lincomycin site without closing the chloramphenicol site, (b) asymmetry of competition effects between chloramphenicol and erythromycin, (c) differential responses to ethanol and (d) differential responses to other antibiotics (puromycin, macrolides, streptogramins A and B and others also acting on the larger ribosome subunit). [ABSTRACT FROM AUTHOR]

1. 30S ribosomal subunits from Escherichia coli were hydrolyzed with ribonuclease T1, and the ribonucleoprotein fragments produced were fractionated on a 5% polyacrylamide/0.5% agarose composite gel. Two main breakdown products were observed corresponding in size to about 20% and 60% of the intact 30S particle. 2. Each fraction from the 5% composite gel was subjected to protein analysis on a periodatesoluble 15% polyacrylamide gel, run in the detergent Sarkosyl. The smaller ribonucleotprotein fragment was found to contain only four of the 30S ribosomal proteins, and these were present in equimolar amounts. The larger fragment had a complex protein composition; it was deficient in the four proteins contained in the smaller fragment, and was considerably enriched in the largest 30S protein. 3. The results show a striking correlation with Nomura's "assembly map" of the 30S particle. [ABSTRACT FROM AUTHOR]

A fraction containing a complex between initiation factors F2 and F3 was isolated from Escherichia coli ribosomes. In contrast to the free factors, this F2 · F3 complex makes a stable ternary association with formylmethionyl-tRNA and GTP. F2 · F3 complex can be formed on the ribosomes during the process of initiation. It acts preferentially on the 30 S ribosomal subunit to which it confers a much larger affinity for fMet-tRNA than observed with the free factors. A model for the function of F2 · F3 complex as an intermediate in the initiation cycle is proposed. [ABSTRACT FROM AUTHOR]

Free N-carbobenzyloxy-phenylalanyl-tRNA is rapidly cleaved by the enzyme peptidyl-tRNA hydrolase. When bound to a 30 S ribosome in the presence of poly(U), the substrate is hydrolyzed as rapidly as when free. The addition of 50 S ribosomal subunits to form the 70 S ribosomal binding complex protects the bound substrate from the enzyme. The degree of protection depends on the state of activity of the 50 S subunit. Under conditions favoring the formation of 70 S ribosomes, active 50 S subunits (capable of catalyzing the peptidyl transferase reaction) afford complete protection at one mole per mole of 30 S subunits Inactive 50 S sub- units also protect the substrate, but less effectively. The protection of bound substrate can be used to measure the amount of 70 S ribosomal binding complex formed and the rate of its formation. Using highly active hydrolase preparations, we have developed a rapid assay procedure and present several examples of its use Under conditions favoring the association of ribosomal subunits, the formation of the 70 S complex may reach completion within a few seconds. [ABSTRACT FROM AUTHOR]

The electrophoretic mobilities of mitochondrial and cell-sap ribosomal RNAs from Saccharomyces carlsbergensis have been measured relative to the mobility of Escherichia coli rRNA in 2.4% polyacrylamide gels at various temperatures and ionic strengths. At 5° C and in 20 sodium acetate, 40 mM Tris-acetate, 2 mM EDTA (pH 7.8), mitochondrial and cell-sap rRNAs co-migrated and were only partially resolved from E. coli rRNA. When the temperature of electrophoresis was increased the mobility of the mitochondrial rRNAs (and to a lesser extent of the cell-sap rRNAs) decreased relative to the mobility of the E. coli rRNA species, reaching a minimum between 9 and 16° C. As a consequence the mitochondrial and cell-sap rRNAs were completely resolved in this buffer at temperatures above 9° C. Similar effects of temperature on relative electrophoretic mobility were observed m 90 mM Tris-borate buffer at pH 8.3 and the effect was even more pronounced at low ionic strength (10 mM NaCl, 5 mM Tris-HCl, 2 mM EDTA, pH 7.5). At low ionic strength the decrease in the relative electrophoretic mobility of mitochondrial rRNA with an increase hi temperature was accompanied by a decrease in sedimentation coefficient from 21-22 S (large component) and 14 S (small component) at 5° C to 16 and 11.5 S at 20° C (calculated m relation to assumed S-values of 23 and 16 S for the rRNA species of E. coli). We conclude that the electrophoretic mobility of RNA in polyacrylamide gels is more dependent on secondary structure than previous work had suggested and that caution is needed to interpret the relative mobility of mitochondrial RNA on gels in terms of molecular weight. In addition, our results show that judicious variation of temperature and ionic strength may allow the separation on gels of RNA mixtures that cannot be resolved under standard conditions. [ABSTRACT FROM AUTHOR]

Chemically synthesized peptidyl-tRNAPhe reacts with puromycin when bound non-enzymatirally to Escherichia coli ribosomes in the presence of poly U. Guanosine triphosphate and G factor have no influence upon the puromycin reaction, when the magnesium ion concentration is 0.01 M or lower, but stimulate the puromycin reaction when the binding of peptidyl-tRNA is carried out at higher magnesium ion concentration. In this stimulation β,γ-methylene-guanosine triphosphate cannot replace GTP, and fusidic acid inhibits the stimulation. The percentage of bound peptidyl-tRNA which reacts with puromycin in the absence of G factor and GTP decreases upon increasing the magnesium ion concentration. From all these observations one can conclude that at magnesium ion concentrations below 0.01 M, peptidyl-tRNA (such as Gly2-Phe-tRNA) binds preferentially to the donor site, but at higher magnesium ion concentrations (such as 0.025 M) the ribosomal acceptor site is preferred. Peptidyl-tRNAPhe can react with puromycin in the absence of the proper mRNA at rather high magnesium ion concentration (0.02 M and higher) but this interaction in the absence of mRNA may be not a common feature of other peptidyl-tRNAs, except tRNAMet. Phe-tRNA interacts with puromycin but the rate of this interaction is much slower than that of peptidyl-tRNAPhe such as Gly2-Phe-tRNA. [ABSTRACT FROM AUTHOR]

A ribosome fraction enriched in "native" subunits by differential centrifugation yields, upon washing in a buffer containing ammonium chloride and magnesium ions, a protein mixture which can dissociate cytoplasmic ribosomes from Saccharomyces cerevisiae into 40 S and 60 S subunits. This heat dependent reaction cannot be related to any marked ribonuclease activity in the protein mixture. Furthermore, the mixture is unable to dissociate Escherichia coli or rabbit reticulocyte ribosomes; this property is unique, since proteolytic enzymes readily dissociate reticulocyte ribosomes as well as yeast ribosomes. Within 30 min of incubation, the factor-dependent dissociation reaches a plateau, due either to the stoichiometric mode of action of the factor, or to the inability of ribosomes to support complete dissociation under the ionic conditions of the experiments. These two points are discussed. [ABSTRACT FROM AUTHOR]

1. A study of sensitivity to T1 ribonuclease of the 16 S and 23 S RNAs within the 30 S and 50 S subunits and the 70 S ribosomes from Escherichia coli has been performed using sucrose gradient sedimentation. 2. The sensitivity of 23 S RNA within the 50 S subunits is greater than that of 16 S RNA within the 30 S subunits. 3. After action of T1 ribonuclease on both 30 S and 50 S subunits, under conditions which would lead to total hydrolysis of the free RNAs, the persistence of protected RNA fragments of large size is observed. 4. A much less significant degradation of 16 S and 23 S RNAs is observed, when T1 ribonuclease action is performed under the same conditions upon the 70 S ribosomes, instead of 30 and 50 S subunits; this supports the existence of regions m both RNAs involved in the association of the two subunits. [ABSTRACT FROM AUTHOR]

Electrophoretic separation in agar gel has been achieved for ribosomes from rat liver and Escherichia coli and their subunits. These particles were characterized by their mobility. RNA was extracted from the agar gel zones containing the two subunits of rat liver. It was found that the faster subunit contained only 18 S RNA with a base composition identical with that of 18 S cytoplasmic rat liver RNA. The slow subunit contained 18 S and 28 S RNA, both having the base composition of 28 S cytoplasmic RNA. The technique also provides a means of studying the labelling pattern of the ribosomes, contaminants being quantitatively removed by washing of the gel with acid. [ABSTRACT FROM AUTHOR]

Poly U-directed stimulation of preincubated calf lens ribosomes revealed that these particles from a mammalian source have a phenylalanine incorporation ability comparable to the Escherichia coli system. A search was made for optimal conditions. After variation of the magnesium concentration during preincubation two levels of maximal incorporation were observed. The region of highest relative stimulation did not coincide with these maxima. Preincubation under conditions of optimal amino acid incorporation yields 80S monomers and to a lesser extent 120S particles. No smaller units or breakdown products were observed in the analytical ultracentrifuge. Such particles could be demonstrated after prolonged dialysis of either polysomes or 80S monomers against 0.1 mM MgC12. Their sedimentation coefficients were 50S and 30S respectively but not 60 and 40 S. [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]

The transfer of valine into haemoglobin from various isoaccepting species of Val-tRNA obtained from Escherichia coli and yeast, which differ in the specificity for codon recognition has been investigated using a rabbit reticulocyte cell-free system. It was demonstrated that the Val-tRNA species which bind to ribosomes in response to the codon GUG preferentially transferred valine into both a and β-globin chains and that the other species of Val-tRNA which cannot repond to GUG were scarcely utilized in the haemoglobin synthesis. Several possibilities to explain this phenomenon were examined and consequently it has been strongly suggested that valine codons in mRNA for rabbit globin, at least for α-globin chain, are very rich in GUG and possibly GUA. [ABSTRACT FROM AUTHOR]

A systematic study has been made of the dissociation into subunits of the ribosomes of Escherichia coli. By analytical ultracentrifugation, ribosomes were shown to be in dynamic reversible equilibrium with their subunits. This enabled a thermodynamic analysis to be made of the association-dissociation reaction and of several factors which influence it: the concentration of monovalent and divalent cations, the presence of transfer RNA and supernatant factors, the pH, and hydrostatic pressure. The apparent change in free energy for the association in 10 mM sodium phosphate pH 7.0 was -17.4 kcal/mol. Association was accompanied by the net binding of magnesium ions or the net release of monovalent cations (potassium, sodium, or Tris ions). Circular dichroism measurements of ribosomes and their isolated subunits over a range of magnesium and potassium ion concentrations indicated that a change in the conformation of each subunit accompanied association. The change occurring in the 50-S subunit was dependent on the presence of potassium ions, whereas the change in the 30-S subunit was magnesium-ion dependent. The conformational change in the 50-S subunit was critical for the association-dissociation reaction. Under ionic conditions which lead to ribosome dissociation one molecule of transfer RNA was released per 70-S ribosome. This was an independent reaction which did not influence the subunit association-dissociation equilibrium. The release of transfer RNA was accompanied by the net release of four magnesium ions, and occurred with an apparent change in the free energy of 20.8 kcal/mol. [ABSTRACT FROM AUTHOR]

1. 30-S ribosomal subparticles were hydrolysed with ribonuclease T1 or pancreatic ribonuclease in the presence of 2 M urea. The RNA · protein fragments produced were separated on 5% polyacrylamide/0.50% agarose composite gels. Fractions from the composite gels were subjected to protein analysis on 17.5% periodate-soluble polyacrylamide gels run in the detergent sarkosyl, using the technique already published. 2. Several RNA · protein fragments were obtained by this procedure, each containing equimolar amounts of a few specific ribosomal proteins. Strict criteria for specificity of the proteins in each fragment were applied. Proteins S8 and S15 were found reproducibly in a small RNA · protein fragment, with only trace amounts of the remaining ribosomal proteins. Six proteins (S7, S9, S10, S13, S14, and S19) constitute another group, and fragments containing three, four, five, or all six of these proteins were observed. Protein S20 was occasionally associated with this group, or with S6, S16 or S17, and possibly S21. The latter (including S20) were also found in association with S8 and S15. 3. The individual proteins found in specific fragments are interpreted as being close neighbours in the 30-S particle. The fragment data can be incorporated into a re-arranged version of Nomura's "assembly map". [ABSTRACT FROM AUTHOR]

The methionine-accepting tRNA species from Escherichia coli, ascites tumor cells, and yeast were tested for their capacity to function in peptide-chain elongation in homologous and heterologous cell-free systems from these organisms comprising polysomes and elongation factors. The following results were obtained 1. The utilization of Met-tRNAfMet in chain elongation is not restricted to mammalian systems but can also be observed in a polysome system from E roll with initiator tRNA from ascites cells and homologous Met-tRNAfMet. 2. In polysome systems from E. coli and ascites tumor cells the magnesium concentrations optimal for the utilization of the homologous methionine-accepting tRNAs in chain elongation were found to he higher than the Mg2+ ion concentrations at which the discrimination between homologous Met-tRNAMet and Met-tRNAfMet is maximal. 3. Of the three systems studied, the polysome system from yeast is characterized by the most stringent exclusion of any initiator tRNA from functioning in peptide-chain-elongation. 4. lnitiator-tRNA from yeast, while recognized by initiation factors from E. coli, is riot utilized for chain elongation in any of the cell-free polysome systems tested. Additional results pertaining to the T factor-induced attachment of all six Met-tRNA species to ribosomes from E. coil, ascites cells and yeast were in complete agreement with these findings. Finally, the influence of a formylation or acetylation of the α-amino group on the recognition of Met-tRNA species from E. coli and ascites tumor cells by fectors T1, Tu,s1, and a crude initiation factor preparation from E. coli was studied. The presence of a free α-amino group could be shown to be an important prerequisite for the interaction of aminoacyl-tRNA and factors T1 or Tu,s. [ABSTRACT FROM AUTHOR]

In an attempt to determine the RNA-protein interactions involved in the biogenesis of the 50-S ribosomal subunit we have developed a simple and specific system in vitro which reflects some of the intricacies of the Escherichia coli 50-S subunit. By studying the interaction of isolated ribosomal proteins with 5-S and 23-S RNAs we have demonstrated the formation of a stable complex which contains 5-S RNA and 23-S RNA and which is dependent upon only two or three ribosomal proteins. The characteristics of this complex have been determined and the responsible proteins identified. The 5-S 23-S RNA complex is dependent upon protein L18 in conjunction with either L6 or L25. Although it would be premature to conclude that these results in vitro are directly related to the assembly map in vivo of the 50-S subunit, there is a strong correlation between our results and those obtained with various natural and artificial ribosomal subparticles. [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]