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The puromycin analogue. 5'-O-(N-bromoacetyl-p-aminophenylphosphoryl)-3'-N-L-phenylalanyl puromycin aminonucleoside, acts as an affinity label in covalently binding to 23-S RNA in the active centre of peptidyl transferase of Escherichia coli ribosomes [Greenwell, P., Harris, R. J. & Symons. R. H. (1974) Eur. J. Biochem. 49, 539-554]. The sequence of a pentanucleotide fragment of 23-S RNA to which the affinity label is attached has now been determined. In an attempt to determine the nucleotide residue to which the affinity label was attached, a 32Plabelled affinity label was reacted with ribosomal RNA, poly(A), poly(C), poly(G) and poly(U) under the conditions used for affinity labelling of ribosomes. However, no specific reaction occurred, confirming our earlier observation that this required the functional integrity of the ribosome. An alternative approach of affinity labelling ribosomes labelled by incubating E. coli in the presence of [³H]adenosine, [³H]cytidine, [³H]guanosine or [³H]uridine, showed that the affinity label was attached to a CMP residue. Effinity-labelled oligonucleotide fragments were purified for sequence analysis as follows. The free α-amino group of the affinity-label molecule attached to 23-S ribosomal [32P]RNA was coupled to biotin by reaction with the N-hydroxysuccinimide ester of biotin. After digestion of the RNA with either ribonuclease A or T1, the basic protein avidin was added to form the avidin, biotin-affinity-label-oligonucleotide fragment. Stepwise chromatography on phosphocellulose, followed (after dissociation of the avidin) by electrophoresis on DEAE-cellulose at pH 1.9 gave substantial purification of the biotin-affinity-label-oligonucleotide. From the nucleotide composition of the purified ribonuclease A and T1 fragments, the pentanucleotide sequence of G-U-C*-C-Gp was obtained, where C* represents C modified by the affinity label. [ABSTRACT FROM AUTHOR]

The kinetics of the puromycin interaction with different ribosomal bound peptidyl-tRNAs were studied. The rates of the puromycin reaction with AA-Phe-tRNA and AA2-Phe-rRNA using three different amino acids (AA) were compared. At 4° aH the three dipeptidyl-tRNAsPhe reacted considerably slower than the three tripeptidyl-tRNAsPhe. Acetyl-Phe-tRNA reacted faster than the dipeptidyl-tRNA, but slower than the tripeptidyl-tRNA. Glyn-Phe-tRNA (n = 3, 6 and 9) reacted with puromycin much faster than Gly-Phe-tRNA and at a rate similar to that of Gly2 Phe-tRNA. Lauroyl-Phe-tRNA reacted with puromycin much slower than acetyl- and octanoyl-Phe-tRNA both at 4° and 30°. [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]

Poly(U)-programmed 70S ribosomes can be shown to be 80% to 100% active in binding the peptidyl-tRNA analogue AcPhe-tRNA to their A or P sites, respectively. Despite this fact, only a fraction of such ribosomes primed with AcPhe-tRNA participate in poly(U)-directed poly(Phe) synthesis (up to 65%) at 14 mM Mg2+ and 160 mM NH+4. Here it is demonstrated that the apparently ‘inactive’ ribosomes (≥ 35%) are able to participate in peptide-bond formation, but lose their nascent peptidyl-tRNA at the stage of Ac(Phe)n-tRNA, with n ≤ 2. The relative loss of early peptidyl-tRNAs is largely independent of the degree of initial saturation with AcPhe-tRNA and is observed in a poly(A) system as well. This observation resolves a current controversy concerning the active fraction of ribosomes. The loss of Ac(Phe)n-tRNA is reduced but still significant if more physiological conditions for Ac(Phe)n synthesis are applied (3 mM Mg2+, 150 mM NH+4, 2 mM spermidine, 0.05 mM spermine). Chloramphenicol (0.1 mM) blocks the puromycin reaction with AcPhe-tRNA as expected but, surprisingly, does not affect the puromycin reaction with Ac(Phe)2-tRNA nor peptide bond formation between AcPhe-tRNA and Phe-tRNA. The drug facilitates the release of Ac(Phe)2–4-tRNA from ribosomes at 14 mM Mg2+ while it hardly affects the overall synthesis of poly(Phe) or poly(Lys). [ABSTRACT FROM AUTHOR]

The puromycin reaction, catalyzed by the ribosomal peptidyltransferase, has been carried out so as to make the definition of two distinct parameters of this reaction possible. These are (a) the final degree of the reaction which gives the proportion of peptidyl (P)-site binding of the donor and (b) the reactivity of the donor substrate expressed as an apparent rate constant (kobs). This kobs varies with the concentration of puromycin; the maximal value (k3) of the kobs, at saturating concentrations of puromycin, gives the reactivity of the donor independently of the concentrations of both the donor and puromycin. k3 is also a measure of the activity of peptidyltransferase expressed as its catalytic rate constant (kcat). If we assume that the puromycin- reactive donor is bound at the ribosomal P site, we observe the following, depending on the conditions of the experiment: the proportion of P-site binding of the donor substrates AcPhe-tRNA or fMet- tRNA can be the same and close to 100%, while there is a tenfold increase in the reactivity of the donor (k3 = 0.8 min-1 versus 8.3 min- 1). On the other hand there are conditions, under which the proportion of P-site binding increases from 30% to 100% while k3 remains low and equal to 0.8 min-1. Using the puromycin reaction it was also found that an increase of Mg2+ from 10 mM to 20 mM reduces the reactivity of the donor and, hence, the activity of peptidyltransferase, provided that this change in Mg2+ occurs during the binding of the donor but not when it occurs during peptide bond formation per se. The fact that the donor substrate may exist in various states of reactivity in this cell-free system raises the possibility that the rate of peptide bond formation may not be uniform during protein synthesis. [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]

Photoactivation of the [³H]dihydrorosaramicin chromophore at a wavelength above 300 nm allows the covalent attachment of the macrolide antibiotic to the bacterial ribosome. Bidimensional electrophoresis shows that the radioactivity is mainly associated with proteins L1, L5, L6, L15, L18, L19, S1, S3, S5, S5 and S9. When photoincorporation of the drug is conducted in the presence of puromycin as effector of [³h]dihydrorosaramicin-binding sites, a decrease in the labeling of most proteins is observed, except for L18 and L19, which are radiolabeled to a larger extent. These results allow us to speculate that L18 and L19 belong to the high-affinity binding site of rosaramicin antibiotic. [ABSTRACT FROM AUTHOR]

We have measured the binding isotherms of C-A-C-C-A(3′NH)-[14C]Phe to the 70S ribosomes and 50S subunits of Escherichia coli and proposed a theoretical model for adsorption when cooperative interaction occurs between ligands that are adsorbed on ribosomes. Analysis of the experimental binding isotherms leads to the following conclusions. A ribosome (or subunit) binds two C-A-C-C-A(3′NH)-Phe molecules. The binding of C-A-C-C-A(3′NH)-Phe to a ribosome (or subunit) is a cooperative process, characterized by a cooperativity coefficient τ = 40 ± 5 or more. The binding of C-A-C-C-A(3′NH)-AcPhe at the donor site of the peptidyltransferase center (association binding constant 1.5 × 106 M-1) and the binding of puromycin at the acceptor site also occur cooperatively with a coefficient of 10–25, the association binding constant of puromycin at the acceptor site being (1–2) × 104 M-1. The puromycin association binding constant at the donor site multiplied by the cooperativity coefficient of two interacting puromycin molecules adsorbed on a ribosome equals 100–200 M-1. [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]

Arginine inhibits the formation of acetylleucyl-puromycin from C(U)-A-C-C-A-LeuAc and puromycin (‘fragment reaction’), catalized by Escherichia coli and yeast ribosomes. From 18 different L-amino acids assayed, arginine was the most effective in producing inhibition (50% inhibition at 20 mM, with 1 mM puromycin). L-Argininamide and D-arginine gave about the same inhibition as L-arginine. The inhibition by L-arginine is competitive with respect to puromycin. The plot of the slopes obtained in a Lineweaver and Burk representation versus [Arg]², and the plot of l/v versus [Arg]² at a fixed concentration of puromycin, are linear, which seems to indicate that two arginine molecules must interact at the puromycin binding site to produce inhibition. In addition to the ‘fragment reaction’, arginine inhibits the non-enzymatic binding of AcPhe-tRNA, C(U)-A-C-C-A-Leu and C(U)-A-C-C-A-LeuAc to ribosomes. However, it does not inhibit poly(U)-directed polyphenylalanine synthesis or the reaction of puromycin with AcPhe-tRNA previously bound to the peptidyl site. The results agree with arginine binding to the acceptor site, and with a sequential mechanism for the ‘fragment reaction’, puromycin binding first. [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]

Ethoxyformic anhydride abolishes the peptidyl transferase activity of 50-S ribosomal subunits, LiCl split proteins and L16. Hydroxylamine treatment results in reactivation. Erythromycin exhibits significant protection with 50-S ribosomal subunits. With LiCl split proteins and L16 significant protection was exhibited only after reconstitution. The results indicate that the ethoxyformic anhydride is reacting with approximately six histidines in LiCl split proteins and one in L16. Since L16 has been reported to contain a single histidine, the results presented indicate the involvement of this histidine in peptidyl transferase activity. [ABSTRACT FROM AUTHOR]

F2 phage RNA treated with O-methylhydroxylamine under denaturing conditions loses its ordered structure with consequent exposure of the normally hidden initiation codons. In the presence of Escherichia coli ribosomes and crude initiation factors modified f2 RNA binds about 50 times more f-[³H]-Met-tRNA than native f2 RNA. The interaction of native f2 [14C]RNA to E. coli 70-S or 30-S ribosomes does not depend on the presence of initiation factors. A significant number of ribosomes deficient in initiation factors interact with a molecule of modified f2 [14C]RNA. Treatment of the resultant polysomal complex with pancreatic Rnase yields ribosomes with f2 RNA fragments protected against Rnase. Almost all AUG/GUG codons in the f2 RNA are located on the Rnse-insensitive ribosome-bound fragments, constituting only 25% of the entire molecule. Addition of crude initiation factors to such ribosomes with fragments of modified f2 RNA promotes binding of f-[³H]Met-tRNA. The resultant complex is fully reactive with puromycin. No binding of Ac-Phe-tRNA takes place under similar conditions. [ABSTRACT FROM AUTHOR]

Micrococcin inhibited the binding of aminoacyl-tRNA at 13 mM Mg2+, but not at 6 mM Mg2+ . The elongation factor Tu (EF-Tu)-dependent binding of aminoacyl-tRNA was inhibited appreciably by 7 μM micrococcin. The EF-G-dependent puromycin reaction of the ribosome-bound aminoacyltRNA, release of unesterified tRNA concomitant with translocation, and the EF-G-dependent evacuation of aminoacyl-tRNA from the acceptor site of the ribosomes were all sensitive to this antibiotic. It is concluded that micrococcin interacts with the acceptor site specifically and this interaction results in the dual action of this antibiotic, namely inhibition of translocation and the binding of aminoacyl-tRNA. [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]

The effect of antibiotics on the coded binding of peptidyl-tRNA to the ribosome and the transfer of the peptidyl residue from peptidyl-tRNA to puromycin has been studied. The coded binding of polylysyl-tRNA was inhibited only by tetracyclines, the binding of acetylphenyl- alanyl-tRNA was inhibited by tetracyclines and streptomycin and stimulated by spiramycin and carbomycin. The most potent inhibitors of the puromycin reaction with polylysyl-tRNA were the macrolides erythromycin, spiramycin, carbomycin and oleandomycin, inhibiting by 50% at a concentration of 0.1 μM; neomycin (1 μM), chloramphenicol (100 μM), tetracyclines (100 μM), streptomycin (50 μM) and streptovitacin A (1 mM) were somewhat less effective. The puromycin reaction with acetylphenylalanyl-tRNA was inhibited by antibiotics in a similar way, only erythromycin stimulated this reaction at concentrations between 0.1 and 500 μM. [ABSTRACT FROM AUTHOR]

Subunits were obtained by incubation of ribosomes with cell sap, puromycin, GTP, phospho-enolpyruvate, pyruvate kinase, subsequent exposure to 0.15 M KCl and 1 mM MgSO4 followed by sucrose density-gradient separation. Addition of ATP reduced the yield of the subunits but had no influence on their purity as judged by polyacrylamide-gel electrophoretic analysis of their RNA. Reassociation of the subunits to form 80-S ribosomes and restoration of polyuridylic-acid-dependent synthesis of polyphenylalanine was highest when the subunits were prepared by incubation of ribosomes in the absence of ATP with or without puromycin. In the absence of puromycin, the dissociation rate per minute was 8% and 2% of the total at 35° and 25°C, respectively. Dietary protein restriction excerted no influence on the dissociation of the ribosomes incubated with cell sap of liver from rats fed a normal protein diet, while cell sap from protein-restricted rats had a decreased capacity to dissociate ribosomes of normal rate. These differences were significant per wet weight of liver but not per mg cell-sap protein, due to a simultaneous decrease in the protein content of the cell. [ABSTRACT FROM AUTHOR]