I. Rychlík

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.

N-Tosyl-L-phenylalanylchloromethane reacts with cysteine 81 in the molecule of elongation factor Tu from Escherichia coli.

Elongation factor EF‐Tu from Escherichia coli was labelled with N‐[14C]tosyl‐L‐phenylalanylchloromethane, digested with trypsin and the peptides obtained separated by HPLC. The only radioactive peak recovered corresponded to tryptic peptide containing residues 75–98. Sequencing of the peptide by automated Edman degradation identified cysteine 81 as the site of N‐tosyl‐L‐phenylalanylchloromethane modification. These results confirm the importance of this residue for the interaction with aminoacyl‐tRNAs.

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.

The metabolism of adenosine 3′,5′-cyclic phosphate. II. Some properties of adenosine-3′,5′-cyclic-phosphate phosphodiesterase from the rat kidney

Abstract 1. The characteristics of adenosine-3′,5′-cyclic-phosphate diesterase have been studied by standard methods and by a newly described isolation technique using the paper chromatographic system isopropanol-NH 4 OH-water (7:2:1, by vol.) followed by n -butanol saturated with water. This latter technique allows all of the metabolites of Ado-3′,5′- P which can arise from mammalian tissue to be separated. 2. Rat-kidney adenosine-3′,5′-cyclic-phosphate phosphodiesterase possesses a number of properties common to the enzyme from other tissues, such as reaction kinetics, pH optimum and methylxanthine and ATP inhibition. F − does not inhibit this enzyme.

The metabolism of adenosine 3′,5′-cyclic phosphate. I. Method for the determination of adenyl cyclase and some properties of the adenyl cyclase isolated from the rat kidney

Abstract 1. 1. A method has been developed for the determination of adenyl cyclase by means of [8-14C]ATP, which is incubated with the enzyme to produce [8-14C]Ado-3′,5′-P. The latter is separated from other products in the reaction mixture by a series of chromatographic systems on paper: isopropanol-NH4OH-water, followed by isopropanol-HCl-water and then n-butanol saturated with water. In this manner Ado-3′,5′-P can be clearly separated from ATP and other metabolites of the latter, no matter what the mammalian tissue source of enzyme. 2. 2. Some of the properties of rat-kidney adenyl cyclase have been studied. The enzyme activity contained in the 600 ×g sediment from the homogenate has a sharp pH maximum about 7.5, is stimulated by F− in the concentration range 3–100 mM.

The binding site for the 3′-terminus of aminoacyl-tRNA in the molecule of elongation factor Tu from Escherichia coli

The mRNA-directed binding of aminoacyl-tRNA to the ribosome requires a special protein factor EF-T, and GTP. A ternary complex between aminoacyl-tRNA, EF-T, and GTP is formed as an intermediate from which the ~~oacyl-t~A is transfe~ed to the ribosomal recognition site (reviewed [ 11). it is the main feature of EF-T, that in the form of EF-T, .GTP it is able to discriminate between aminoacylated and nonor acylaminoacyfated tRNAs [2-4]. This provided a basis for the accumulat~g evidence that the 3’-terminus of aminoacyl-tRNA is involved in the recognition reaction between aminoacyl-tRNA and the elongation factor T, [S-7]. Therefore, we prepared two analogs of the 3’oterminus of phenylalanyl-tRNA, 2’(3’)0L-phenylalanyladenosine and cytidylyl-(3’ + 5’~2’(3’)U-L-p~enylalanyladenosine and tested them for the ability to replace aminoacyl-tRNA in protecting the aminoacyltRNA binding site of EF-T, from inactivation by N-tosyl-L-phenylalanylchloromethane. The results presented here show that: (1) A-Phe and CpA-Phe can interact with the aminoacyl-tRNA binding site of EF-T,; (2) The SH group in the aminoacyl-tRNA binding

Chemical evidence for the involvement of histidyl residues in the functioning of Escherichia coli elongation factor Tu

Peptide chain elongation factor Tu (EF-Tu) catalyses GTPdependent binding of ~oacyl-tea to the A site of ribosomes through the intermediate formation of a ternary EF-Tu . GTP a aminoacyltRNA complex (reviewed [l]). We have tried [2,3] to identify the regions in both EF-Tu and the aminoacyl-tRNA involved in their mutual bin~ng and proposed a model of the interaction between EF-Tu . GTP and aminoacyl-tRNA. The model implies that the 3’-terminus of the aminoacyltRNA interacts with EF-Tu, and that the SH-group of cysteine or/and some ammo acid residue(s) Iocated close to this cysteine in the aminoacyl-tRNA binding site of the factor could be involved in the ~teraction with the 3’“terminus of aminoacyl-tRNA [3]. The observation that the tryptic peptide of E. coli EF-Tu containing the cysteine residue spoilt for aminoacyl-tRNA binding is rich in hi&dine residues and particularly the fact that two of these residues are located ~mmetric~y around the cysteine [4] led us to investigate the role of histidine residues in the function of EF-Tu. Here we describe studies on the inactivation of EF-Tu . GDP by diethylpyrocarbonate, a relatively specific histidine reagent [5], and by photooxidation in the presence of the rose bengal dye which is the most selective method available for the modification of histidine residues in proteins [6]. The results of these experiments and the amino acid analysis of the photooxidized factor suggest that histidine residues are involved in the bind~g of EF-Tu to aminoacyltRNA and/or ribosomes. 2. Materials and methods

Tosylphenylalanyl chloromethane-inhibitor of complex of S1S3-factors in cell-free protein-synthetizing system from Bacillus stearothermophilus

The elongation of the growing peptide chain during its biosynthesis on the ribosome represents a complicated process involving the participation of several protein factors contained in the S-100 supernatant fraction. The mechanism of their action is at present intensively investigated [ 1, 21 . This study provides evidence showing that tosylphenylalanyl chloromethane (TPCK, 1-chloro-4-phenyl-3tosyl-amido-2-butanone) is a selective irreversible inhibitor of the complex of Sr S3 -factors in the cellfree protein-synthetizing system from B. sfearothermophilus. As reported elsewhere, TPCK inhibits irreversibly also the T-factor from E. coli [3] .

Mode of action of N-tosyl-L-phenylalanylchloromethane on the elongation protein-synthesizing S 3 factor from Bacillus stearothermophilus.

Abstract Preincubation of combined elongation factors S1 + S3 (the S1S3 complex) from Bacillus stearothermophilus with N- tosyl- l -phenylalanylchloromethane results in a loss of their ability to stimulate the binding of phenylalanyl-tRNA to the complex of acetylphenylalanyl-tRNA-ribosome-poly(U) and of the ability to stimulate the synthesis of polyphenylalanine directed by poly(U) in a cell-free system. The inhibitory effect of N- tosyl- l -phenylalanylchloromethane on the S1S3 complex is substantially increased by an addition of GDP. The S1S3 complex can be also inhibited by p-chloromercuribenzoate. The inhibition is reversible since dithiothreitol restores more than 80 % of the original activity. The product formed after the reaction of the S1S3 complex with p-chloromercuribenzoate is completely insensitive to the usual inactivating effect of N- tosyl- l -phenylalanylchloromethane whether with or without GDP. Hence it is assumed that the inhibitory effect of N- tosyl- l -phenylalanylchloromethane is caused by an interference of the inhibitor with the sulfhydryl group(s) of the enzyme complex which are essential for its activity. There is indirect evidence that the actual component of the S1S3 complex thus affected is the S3 factor (aminoacyl-tRNA-binding factor).