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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]

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 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]

Binding of the yeast Tyr-tRNA and Phe-tRNA to the A site, and the binding of their acetyl derivatives to the P site of poly(U11,A)-programmed Escherichia coli ribosomes was studied. Spermine stimulated the rate of binding of both tRNAs at least threefold, enabling more than 90% final saturation of both ribosomal binding sites. The effect is observed when the tRNAs, but not ribosomes or poly(U11,A), are preincubated with polyamine. Regardless of the binding site, optimal saturation was reached at spermine/tRNA molar ratios of 3 for tRNAPhe and 5 for tRNATyr. The same low spermine/tRNA ratios were previously reported to stabilize the conformation of these tRNAs in solution. On the other hand, the messenger-free, EF-Tu- and EF-G-dependent polymerization of lysine from E. coli Lys-tRNA is drastically reduced, while the poly(A)-directed polymerization is stimulated by spermine through a wide range of Mg2+ concentrations. Misreading of UUU codons as isoleucine, assayed by the A-site binding of E. coli Ile-tRNA, is also inhibited by spermine. All these results demonstrate that spermine increases the efficiency and accuracy of a series of macromolecular interactions leading to the correct incorporation of an amino acid into protein, at the same time preventing some unspecific or erroneous interactions. From the analogy with its known structural effects, it can be inferred that spermine does so by conferring on the tRNA a specific biologically functional conformation. [ABSTRACT FROM AUTHOR]

A and P sites of Escherichia coli ribosomes were titrated with AePhe-tRNAPhe, in the absence or presence of tetracycline. The P-site location of the bound AcPhe-tRNA was assessed by means of a quantitative puromycin reaction. The results demonstrate that, in agreement with the generally held view, tetracycline exclusively inhibits the A-site binding, if the statistical number of bound acyl-tRNA molecules per ribosome does not exceed about 0.5. However: above this value the P site becomes sensitive to tetracycline as well. It follows that the tightly coupled 70S ribosomes used in functional studies appear to be functionally heterogeneous, i.e. those P sites which cannot be affected by tetracycline are preferentially occupied by AcPhe-tRNA, whereas higher concentrations of this tRNA species are required to fill tetracycline-sensitive P sites. Furthermore, the results imply that under tRNA saturation conditions the tetracycline inhibition cannot be used as an indicator for the site location of bound tRNA. [ABSTRACT FROM AUTHOR]

The standard technique for determination of the ribosomal site location of bound tRNA, viz. the puromycin reaction, has been analyzed with regard to its applicability under tRNA saturation conditions. The criteria derived have been used to re-examine the exclusion principle for peptidyl-tRNA binding, which states that only one peptidyl-tRNA (AcPhe-tRNA) can be bound per ribosome although in principle two sites (A and P site) are available. The following results were obtained. 1. The puromycin reaction is only appropriate for a site determination if the reaction conditions prevent one ribosome from performing more than one puromycin reaction. With an excess of AcPhe-tRNA over ribosomes, and in the absence of EF-G, this criterion is fulfilled at 0 °C, where the P-site-bound material reacts with puromycin (quantitative reaction after 50 h), while the A-site-bound material does not. In contrast, at 37°C the extent of the puromycin reaction can exceed the binding values by 2&ndashh;4-fold (‘repetitive reaction’). In the presence of EF-G a repetitive puromycin reaction is seen even at 0 °C, i.e. EF-G can already promote a translocation reaction at 0 °C. However, the extent of translocation becomes negligibly low for short incubation times (up to 60 min) at 0 °C, if only catalytic amounts of EF-G are used. 2. Using the criteria outlined above, the validity of the exclusion principle for Escherichia coli ribosomes was confirmed pursuing two different experimental strategies. a) Ribosomes were saturated with AcPhe-tRNA at one molecule per 70S ribosome, and a quantitative puromycin reaction demonstrated the exclusive P-site location of the AcPhe-tRNA. The same result was also found in the presence of viomycin, which blocks the translocation reaction. These findings also indicate that here nearly 100% of the ribosomes participate in AcPhe-tRNA binding to the P site. b) Precharging the P sites of 70S ribosomes with one Ac[14C]Phe-tRNA molecule per ribosome prevented additional Ac[³H]Phe-tRNA binding. In contrast, 70S particles carrying one molecule of [14C]tRNAPhe per ribosome were able to bind up to a further 0.64 molecule Ac[³H]Phe-tRNA per ribosome. [ABSTRACT FROM AUTHOR]

Escherichia coli 70-S ribosomes contain a third site for tRNA binding, additional to the A and P sites. This conclusion is based on several findings. Direct measurements showed that in the presence of poly(U), when both A and P sites are occupied by Ac[14C]Phe-tRNAPhe, ribosomes are capable of binding additionally deacylated non-cognate [³H]tRNA. If ribosomes in the preparation are active enough, the total binding of labeled ligands amounted to 2.5 mol/mol ribosomes. In the absence of poly(U), when the A site can not bind, the P site and the ‘additional’ site can be filled simultaneously with Ac[14C]Phe-tRNAPhe and deacylated [³H]tRNA, or with [³H]tRNA alone; the total binding exceeds in this case 1.5 mol/mol ribosomes. The binding at the ‘additional’ site is not sensitive to the template. [³H]tRNA bound there is able to exchange rapidly for unlabeled tRNA in solution. Deacylated tRNA is prefered to the aminoacylated one. The binding of AcPhe-tRNAPhe was not observed there at all. The 3′-end adenosine is essential for the affinity. The function of the ‘additional’ site is not known, but its existence has to be considered when tRNA · ribosome complexes are studied. [ABSTRACT FROM AUTHOR]

Analysis of various RNA species from Escherichia coli. The RNAs were prepared under mild conditions without spreading using the shadow-cast technique. The 16-S RNA looks like a flattened balloon of diameter 18 ± 1.9 nm with a smooth surface and regular contours. In contrast, structural domains can be detected in 23-S RNA; typically a central domain is surrounded by five to seven satellite domains, with an overall diameter of 33 ±_ 4.5 nm. Different 23-S RNA preparations, which also behave differently in reconstitution assays, differ with respect to extent and height of the satellite structure. 5-S RNA shows an extended rod-like structure with a length of 11 ± 1.6 nm. A more corpuscular and prominent structure was observed for tRNA, with diameters Phe of 7.7 ± 1.1 nm and 8.9 ± 1.0 nm in case of tRNAPhe, and N-acetylphenylalanyl-tRNAPhe respectively. Analysis of the assembly in vitro of the ribosomal 50-S subunit. The assembly of the 50-S subunit in vitro occurs via three reconstitution intermediates, which are formed subsequently during the first incubation of the two-step reconstitution procedure [Dohme, F. and Nierhaus, K. H. (1976) J. Mol. Biol. 107, 585-599]. A total reconstitution was performed, aliquots were withdrawn during the first incubation at various times and analyzed in the electron microscope. A second aliquot was taken at the same times, subjected to a standard second incubation and tested for poly-(Phe)-synthesizing activity in the presence of 30-S subunits. The activity obtained is a measure of the amount of (second plus third) reconsitution intermediates present in the aliquot. With this parameter it was possible to select a series of subsequent assembly states from the electron micrographs which cinematographically illustrate the assembly of the 50-S subunit in vitro. The results indicate that the reconstitution intermediate stage RI[IPQ]50(1) comprises various particles, which evidently are formed consecutively. In contrast, the following reconstitution intermediate stage RI50(2) appears as a homogeneous population of particles. [ABSTRACT FROM AUTHOR]

fMet-tRNAfMet binds reversibly to the donor site (P-site) of Escherichia coli ribosomes both in the absence of messenger and in the presence of ApUpGp and some other oligonucleotides or poly(U). Kinetics of interaction conforms the second-order law. The equilibrium constants and the rate constants of binding are estimated at 0°C. Not only the cognate trinucleotide ApUpGp but also some other oligonucleotides and even poly(U) stimulate the binding. The presence of total deacylated tRNA considerably increases the selectivity of association. [ABSTRACT FROM AUTHOR]

The poly(U)-dependent binding of Tyr-tRNATyr to Escherichia coli ribosomes has been studied using a highly purified system. Binding is maximal at 10 mM magnesium acetate (up to 0.7 molecule Tyr-tRNATyr/ribosome), and requires the presence of elongation factor (EF) T (a mixture of EF-Ts and EF-Tu), GTP, NH4+ ions and an aminoglycoside antibiotic (streptomycin, neomycin B, kanamycip B or gentamicin C1a). Under limiting and up to saturating concentrations of EF-T, one molecule of GTP is hydrolyzed per molecule of Tyr-tRNATyr bound, suggesting that ‘proof-reading’ mechanisms involving the hydrolysis of GTP are inoperative in the presence of the antibiotics. Binding of Tyr-tRNATyr apparently takes place at the ribosomal acceptor site, since peptide bonds are readily formed with N-acetyl-Phe-tRNA prebound to the ribosomal donor site. In contrast to Phe-tRNAPhe binding, Tyr-tRNATyr binding is impaired by the omission of the 50-S subunit, the replacement of GTP by its non-hydrolyzable analogs guanyl-5′-yl methylene diphosphonate and guanyl-5′-yl iminodiphosphonate, and also by the presence of the antibiotic streptogramin A. This suggests that the correct interaction of Tyr-tRNATyr with the peptidyl transferase centre is essential for the stability of this ligand on the ribosome. Moreover, the aminoglycoside antibiotics are also necessary, even after the binding reaction is complete, to maintain Tyr-tRNATyr on the ribosome. [ABSTRACT FROM AUTHOR]

The metabolism of guanosine 3′,5′-bis(diphosphate) (ppGpp) has been studied in a number of different microbial species including Escherichia coli, Bacillus stearothermophilus, Bacillus subtilis and Bacillus brevis. The results can be summarized as follows. 1. Generally, in the E. coli and B. subtilis systems, a stringent factor catalyses the ppGpp-synthesizing reaction only when activated by uncharged tRNA codon-specifically bound to ribosomes. In the B. stearothermophilus and B. brevis systems the stringent factor does not require activation by ribosomes. The physiological significance of this is reflected by the subcellular distribution: stringent factors requiring ribosomes for activity are exclusively found in ribosomal fractions, while those functioning independently of ribosomes are preferentially present in the high-speed supernatant fraction. Formation of ppGpp is quite specific in that neither ATP as pyrophosphate donor nor GDP (or GTP) as acceptor can be replaced by other nucleotides. However some nucleo- tides, particularly ppApp, effectively inhibit ppGpp production, while others, e.g. ppGpp or A(5′)p4(5′)A, are not inhibitory. 2. Degradation of ppGpp is catalyzed by a guanosine 3′,5′-bis(diphosphate) 3′-pyrophospho-hydrolase present in the ribosomal fraction which specifically releases pyrophosphate from the 3′ position of ppGpp. The activity is apparently associated with ribosomes and, unlike stringent factor, is not completely released under low-ionic-strength conditions. The enzyme has a molecular weight of 65000–70000 and forms dimers and tetramers having molecular weights of 130000 and 260000, respectively. It can also be released from ribosomes by 1 M potassium acetate extraction but then is insoluble in buffers of low ionic strength except in the presence of urea. It is inhibited by tRNA regardless of whether or not ribosomes are present. [ABSTRACT FROM AUTHOR]

Formylmethionyl-tRNA has been found to bind to Escherichia coli ribosomal protein SI. Com- plexes of S1 and methionine-labeled fMet-tRNAfMet were detected by binding to cellulose nitrate membranes. Complex formation was stimulated by incubation at low ionic strength, at 37°C and in the absence of Mg2+. Protein S1 also bound [3H]poly(rC) under the same conditions or in buffers of higher ionic strength, 10 mM in Mg2+, and during incubation at 0°C. Unlabeled poly(rC) was an effective inhibitor of the binding of labeled fMet-tRNA when both nucleotides were added simultaneously to the reaction mixtures; however, approximately 80% of previously bound f[35S]Met-tRNA remained bound after the subsequent addition of unlabeled poly(rC). Binding of f[3sS]Met- tRNA, but not the binding of [3H]poly(rC), was inhibited by polyamines or aminoglycoside antibiotics. Protein S1 that had been reacted with N-ethylmaleimide failed to bind f[3sS]Met-tRNA but was still capable of binding [3H]poly(rC). The data support the concept of two distinct RNA-binding sites on the S1 molecule. Initiator tRNA appears to occupy a site concerned with the RNA unwinding property of S1, whereas poly(rC) binds primarily at a separate site. During initiation on the ribosome, the poly(rC) site may interact with the 3′ end of 16-S ribosomal RNA, while the other site may interact with either mRNA or fMet-tRNA. [ABSTRACT FROM AUTHOR]

Binding of acetyl or formyltryptophanyl-tRNATrp from Escherichia coli or beef liver to E. coli ribosomes is strongly stimulated by E. coli initiation factors and requires GTP. The N-acylated tryptophan is puromycin reactive. Polypeptide chain initiation with acetyltryptophan dependent on poly(U,G) has been demonstrated and is highly dependent on added initiation factors, tRNATrp appears, therefore, to share some structural features with tRNAfMet of significance to the process of polypeptide chain initiation. [ABSTRACT FROM AUTHOR]

It was shown that at 20 mM Mg2+ and 0 or 20 °C each 70-S ribosome binds non-enzymatically about one Phe-tRNAPhe molecule in the A site if poly(U) is used as messenger. Only a minor amount of ribosomes (never exceeding 15%) and after long incubation binds a second molecule of Phe-tRNAPhe and forms diphenylalanyl-tRNA. If the Phe-tRNAPhe preparation contains nonspecific deacylated tRNA, the latter is always bound in the P site up to one molecule per 70-S ribosome. The association constant for this non-specific tRNA binding is equal to (1.4 ± 0.3) × 107 M-1 and is independent of the messenger poly(U) and the filling of the A site by a specific aminoacyl-tRNA. It was found that the codon-dependent binding of Phe-tRNAPhe in the A site is reversible, but two different types of complexes are formed with drastic differences of binding constants. This heterogeneity is due to the presence of two conformers of Phe-tRNAPhe. The equilibrium of the two Phe-tRNAPhe conformers depends on Mg2+ and can be shifted if tRNA is heated and Mg2+ added or sequestered by complexing agents. One type of Phe-tRNAPhe yields complexes with 70-S ribosomes showing extremely high affinity constants (higher than 1011 M-1). The second conformer is characterized by a comparatively low binding constant about 5 × 106 M-1. In the binding of both species to 30-S subunits no difference is seen. A big difference in the behaviour of both Phe-tRNAPhe conformers is found also in the absence of messenger RNA. The association constant of Phe-tRNAPhe (high-affinity conformer) to 70-S ribosomes is about 3 × 108 M-1, but for the low-affinity conformer is hundred times lower. [ABSTRACT FROM AUTHOR]

30-S ribosomal subunits are inactivated by iodination for both enzymic fMet-tRNA and non- enzymic Phe-tRNA binding activities. This inactivation is due to modification of the protein moiety of the ribosome. Reconstitutions were performed with 16-S RNA and mixtures of total protein isolated from modified subunits and purified proteins isolated from unmodified subunits. This allowed identification of the individual proteins which restore tRNA binding activity. S3, S14 and S19 were identified as proteins involved in fMet-tRNA binding. S1, S2, S3, S14 and S19 were identified as proteins involved in Phe-tRNA binding. Modified particles show normal sedimentation constants and complete protein compositions both before and after reconstitution. This suggests that the loss of activity is due to modification of one or more of the actual binding sites located on the 30-S subunit and that restoration of activity is due to structural correction at this site rather than to correction of an assembly defect. [ABSTRACT FROM AUTHOR]

Titration of isolated Escherichia coli ribosomal 30-S particles with [14C]phenylalanyl-tRNA in the presence of poly(uridylic acid) was used for a quantitative assay of codon-dependent binding of aminoacyl-tRNA with the small ribosomal subparticle. The technique has allowed the estimation both of the fraction of "active" 30-S subparticles capable of forming the 30-S · poly(U) · phenylalanyl- tRNA complexes and the equilibrium constants of phenylalanyl-tRNA binding in different media. Heterogeneity of the ternary complexes formed has been revealed: at least two classes of complexes differing in stability have been observed. The stability of the 30-S · poly(U) · phenylalanyl-tRNA complexes has been shown to decrease with the lowering of the Mg2+ concentration, the increase of K+ concentration and the addition of urea. The stability of the complexes increases with the increase of Mg2+ concentration, with the addition of ethanol and decrease of temperature. It is demonstrated that the fraction of actively binding 30-S particles also varies in different medium conditions; it decreases with the increase of ionic strength (K+) and with the addition of urea, and increases with the increase of Mg2+ concentration and addition of ethanol. [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]

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 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]

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]

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]