J. Černá

Substrate specificity of ribosomal peptidyl transferase: 2′(3′)-O-aminoacyl nucleosides as acceptors of the peptide chain on the amino acid site☆

Abstract Synthetic substrates were used to investigate the specificity of the acceptor site of ribosomal peptidyl transferase. The results may be summarized as follows: 1. (i) Simple synthetic compounds, namely 2′(3′)- O -aminoacyl-adenosines representing the ultimate terminal residue of aminoacyl-tRNA, can replace aminoacyl-tRNA in the transfer reaction and serve as acceptors of the nascent peptide residue, both with AcPhe-tRNA ‡ and with (Lys) n -tRNA as the peptide donor. 2. (ii) The acceptor activity of the substrates is influenced by the nature of the side chain of the amino acid residue bound to adenosine; A-Phe was nearly as active as puromycin while A-Gly and A > (CH 2 NH 2 ). (OEt) were inactive. 3. (iii) For an association between ribosomal peptidyl-transferase and the acceptor substrates the presence of free 2′-OH group in the ribose moiety of aminoacyl-adenosines is required as is shown by the low activity of dA-Phe in comparison with A-Phe. 4. (iv) The acceptor activity of the substrates tested is specific with respect to the nucleoside to which the amino acid residue is bound. Activity decreased in the sequence puromycin, A-Phe, I-Phe, C-Phe; G-Phe and U-Phe did not serve as acceptors of the peptide chain. Using AcPhe-tRNA as the donor of the AcPhe residue and A-Phe, C-Phe and I-Phe as acceptors, we isolated A-PhePheAc, C-PhePheAc and I-PhePheAc as terminal products of the reaction. The individual building blocks of aminoacylribonucleosides tested as such did not act as acceptors. Negative results were obtained with ammonium ions, tyramine, free amino acids, their amides and esters, adenosine, inosine and the puromycin aminonucleoside.

Effects of macrolide antibiotics on the ribosomal peptidyl transferase in cell-free systems derived from Escherichia coli B and erythromycin-resistant mutant of Escherichia coli B

Abstract The effect of macrolide antibiotics on the formation of the peptide bond by ribosomal peptidyl transferase was compared in a normal and in an erythromycin-resistant strain of Escherichia coli B. The formation of the peptide bond was studied in simple ribosomal systems where the donors of the acylaminoacyl residue were (Lys) n -tRNA, acPhe-tRNA and terminal acLeu-pentanucleotide and acPhe-pentanucleotide; as acceptors of the acylaminoacyl residue we used puromycin and A-(Phe). In ribosomes of the strain sensitive to erythromycin, other macrolide antibiotics affect the transfer of the acylaminoacyl residue to puromycin, catalyzed by peptidyl transferase. The effect of the antibiotic depends on the nature of the transferred acylaminoacyl residue. The transfer of lysine peptides is inhibited by all macrolide antibiotics tested here: erythromycin, oleandomycin, spiramycin and carbomycin. The transfer of the acPhe- and acLeu- residues is inhibited by spiramycin and carbomycin but it is markedly stimulated by erythromycin and oleandomycin. The inhibition of the transfer of acLeu- or acPhe- residues by spiramycin or carbomycin can be reversed by erythromycin. The effect of macrolides is not substantially affected by whether the donor of the acylaminoacyl residue is formed by an intact molecule of tRNA or only by its terminal fragment. Ribosomes isolated from E. coli B resistant to erythromycin are resistant to the effect of erythromycin and display cross resistance to spiramycin, carbomycin, oleandomycin and chloramphenicol. Cross resistance to chloramphenicol was not observed in the fragment reaction. Macrolide antibiotics display a weaker effect on ribosomes resistant to erythromycin than on ribosomes from a sensitive strain. This holds both for activating and for inhibiting macrolides. Hybridization experiments with 30-S and 50-S ribosomal subunits from a parent and from a resistant strain showed that the 50-S subunit is the resistant component. This was also shown by a fragment reaction with only the 50-S subunit.

Substrate specificity of ribosomal peptidyl transferase. II. 2′(3′)-O-Aminoacyl nucleosides as acceptors of the peptide chain in the fragment reaction

Abstract Transfer of the acetyl- l -leucine (AcLeu) residue from AcLeu-pentanucleotide to synthetic substrates under conditions of the fragment reaction was used to study the specificity of the acceptor site of ribosomal peptidyl transferase. 2′(3′)-O-Aminoacyl nucleosides are the simplest acceptor substrates. Their acceptor activity is dependent on the nature of the nucleoside to which the amino acid residue is bound. The acceptor activity decreased in the sequence 2′(3′)-O- l -phenylalanyladenosine (A-(Phe)) > I-(Phe) > C-(Phe); U-(Phe) was inactive. The presence of a free 2′-hydroxyl group in the ribose moiety of the aminoacyl adenosines was important for the acceptor activity, as shown by the low activity of dA-(Phe) in comparison with A-(Phe). Acceptor activity was influenced by the nature of the side chain of the amino acid residue bound to adenosine: A-(Phe) was a more active acceptor than puromycin, while the acceptor activity of 2′(3′)-O-glycyladenosine A-(Gly) was very low. Free phenylalanine, phenylalanine methyl ester, and adenosine did not act as acceptors. As terminal products of the reactions of AcLeu-pentanucleotide with puromycin, A-(Phe), I-(Phe) and C-(Phe), we isolated AcLeu-puromycin, 2′(3′)-O- acetyl- l -leucyl- l -phenylalanyladenosine (A-(AcLeu-Phe)), I-(AcLeu-Phe) and C-(AcLeu-Phe), respectively.

Effect of cytidine-5′-monophosphate on peptidyl transferase activity

The transfer reaction with pA-fMet as a donor substrate is strongly stimulated by CMP, whereas the transfer reaction with CpApCpCpA-acLeu as a donor substrate is inhibited by CMP. These results indicate that the donor site of peptidyl transferase contains specific binding sites for the terminal adenosine and for the cytidylic acid residue in the terminal sequence CpCpA of tRNA and that an attachment of proper nucleotides to the donor site induces a conformational change in peptidyl transferase.

The effect of gougerotin analogues on ribosomal peptidyl transferase.

1. Introduction Gougerotin (I), a dipeptidyl pyrimidine nucleoside antibiotic, has been found to specifically inhibit the step of protein biosynthesis which is catalyzed by ribosomal peptidyl transferase [l-S] . Since three gougerotin analogues have been synthesized recently [6] , 1-[3(sarcosyl-D-seryl-)amino3-deoxy

Phenylboric acids — a new group of peptidyl transferase inhibitors

-D-gluco- pyranosyl-)uracil (II, cf. fig. l), I-[3-(sarcosyl-D- seryl-)amino-3-deoxy-P-D-glucopyranosyl-)cytosine (III) and l-[4-(sarcosyl-D-seryl-)amino-4-deoxy-O-D- glucopyranosyl-)cytosine (IV), we made an attempt to characterize the mechanism of action of gougerotin (I) and its analogues (II-IV) on the activity of ribo- somal peptidyl transferase. The results are presented below, together with a discussion of relationships be- tween structures I-IV and their inhibitory activity. 2. Materials and methods Ribosomes were prepared from

The effect of antibiotics on the substrate binding to the acceptor and donor site of ribosomal peptidyltransferase of an erythromycin-resistant mutant of Escherichia coli

Inhibitors of peptidyl transferase studied so far have been mainly either antibiotics or structural analogues of part of the natural substrate. In this paper we present evidence that a different group of compounds, namely phenylboric acid and its derivatives, inhibits peptidyl transferase rather strongly. Phenylboric acid has been hitherto’known as a specific inhibitor of serine proteases [ 1 ] which acts as a transition-state analogue by forming a tetrahedral adduct with the serine -OH group and the histidine residue in the catalytic centre of these proteases [2]. Phenylboric acid also forms a complex with cis-diol groups, i.e., the 2’,3’-cis-diol group of ribose at the 3’-terminus of RNA [3]. In this paper we describe the effect of phenylboric acid on the catalytic and binding properties of peptidy1 transferase of Escherichia coli ribosomes.

Inactivation and reactivation of the acceptor binding site of ribosomal peptidyl transferase

Abstract Antibiotics inhibiting peptidyltransferase have been classified into three groups according to their action on the substrate binding to the acceptor and donor site of ribosomal peptidyltransferase. 1. Group 1: erythromycin and oleandomycin stimulate the binding of both the acceptor and donor substrate. These antibiotics have a weaker stimulating effect on the binding to the donor site and stronger one on the binding to the acceptor site of ribosomes from an erythromycin-resistant mutant than from a sensitive strain. 2. Group 2: spiramycin, carbomycin and lincomycin inhibit the binding of both the acceptor and donor substrate. The binding of both substrates to the ribosomes from the erythromycin-resistant strain is less inhibited than the binding to the ribosomes from the parent strain. 3. Group 3: gougerotin and chloramphenicol stimulate the binding of the donor substrate and inhibit the binding of the acceptor substrate. The binding of the acceptor substrate to erythromycin-resistant ribosomes is inhibited by chloramphenicol to the same extent, whereas the inhibition by gougerotin is weaker; on the other hand, the binding of the donor substrate is less stimulated by chloramphenicol and more so by gougerotin. The antagonistic effect of erythromycin on the inhibition of the binding to the acceptor and donor sites by other antibiotics has been examined with sensitive and erythromycin-resistant ribosomes. Erythromycin completely abolishes the inhibition of the binding of both acceptor and donor substrates to sensitive ribosomes caused by spiramycin, carbomycin and lincomycin, and reverses about 50 % of the inhibition of the binding to the acceptor site caused by chloramphenicol, gougerotin and puromycin. In resistant ribosomes, erythromycin affects the inhibition of the binding caused by the aforesaid antibiotics in the same manner as in sensitive ribosomes, except for the inhibition of the binding to the acceptor site by gougerotin, which is not affected by erythromycin at all.

Stimulatory effect of cycloheximide and related glutarimide antibiotics on liver uridine kinase

The ribosomal 50 S subunit may assume two conformations differing in biological activity [ 1, 21 . In one conformation of the 50 S subunit peptidyl transferase (which is a component of this subunit) catalyzes the transfer of the peptide chain from peptidyl-tRNA to puromycin. In the second conformation the enzyme is inactive. The enzymically active conformation is converted to the inactive conformation by removal of NH: ions from the medium. The biologically inactive conformation can be converted back to the active one upon incubation with ammonium ions at a higher temperature (40’) [ 1,2] . The reactivation is markedly temperature-dependent so that practically no reactivation takes place at 0” even in the presence of ammonium ions. A similar temperature dependence was observed by Nomura and Erdmann for the reconstitution of the 50 S subunit from its components ]31 . The inactivation of peptidyl transferase which takes place during the change of ribosome conformation may be caused by a decrease of affinity for the corresponding substrate at the donor site, the acceptor site or at both simultaneously. In addition the change in conformation of the 50 S subunit may result in a distortion of the spatial arrangement of the binding sites so that no peptide transfer can take place even if both binding sites are occupied by the proper substrates. In this paper the inactivation mechanisms were studied. 2. Methods

Photoinactivation of peptidyl transferase binding sites

Cycloheximide inhibits protein synthesis in a variety of mammalian cells, including hepatocytes, Lcells, and reticulocytes [l] . The drug prevents the transfer of amino acids from aminoacyl-tRNA to the growing polypeptide chain [2] and evidence has been obtained showing that cycloheximide inhibits both peptide initiation and extension by an effect on the donor site on ribosomes [3]. After the administration of cycloheximide the formation of ribosomes and the synthesis of nuclear 45 S RNA in rat liver are also decreased as a result of the reduced protein synthesis [4]. The mechanism of cycloheximide action is influenced by adrenal secretion [S] ; in adrenalectomized rats no inhibition of amino acid incorporation into liver proteins has been observed [6]. Besides inhibiting protein synthesis cycloheximide increases tyrosine aminotransferase activity [7, 81. It has been concluded that degradation as well as synthesis of the enzyme must be blocked in the cycloheximide-treated animals [9]. In the present report evidence is presented showing that another liver enzyme, uridine kinase, is influenced by the administration of cycloheximide and related glutarimide antibiotics.