Jiří Jonák

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.

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).

In activation of protein-synthesizing T-factor by N-Tosyl-l-phenylalanyl chloromethane

Abstract N- Tosyl- l -phenylalanyl chloromethane causes the irreversible inhibition of protein synthesis in poly(U)- and poly(A)-directed cell-free systems from Escherichia coli. The elongation factor T is the component affected by the inhibitor. The T-factor inactivated by the chloromethylketone loses its ability to stimulate the binding of aminoacyl-tRNA to ribosomes. Activities of aminoacyl-tRNA ligases, ribosomes and the elongation factor G are not altered.

Role of messenger RNA in binding of peptidyl transfer RNA to 30-S and 50-S ribosomal subunits

The character of oligolysyl-tRNA binding to ribosomal subunits of Escherichia coli A19 was studied. n nThe oligolysyl-tRNA binding to both the 30-S and the 50-S subunits was stimulated by poly A. Poly U and poly (U, C) did not affect the oligolysyl-tRNA interaction with the 50-S subunit; they decreased, however, the basal interaction of oligolysyl-tRNA with the 30-S subunit. n nThe coded oligolysyl-tRNA binding to the 30-S subunit took place slowly and the maximum was attained in 20 min at 35°. By contrast, the oligolysyl-tRNA binding to the 50-S subunit was completed in less than 1 min at 0–35°. n nNH4+ and Mg2+ were essential for the oligolysyl-tRNA binding to the 30-S subunit. Tris+ at a concentration higher than 0.01 M considerably inhibited this coded interaction. Tetracycline at a concentration of 0.6 mM inhibited almost completely the coded interaction of oligolysyl-tRNA with the 30-S subunit. n nThe coded oligolysyl-tRNA binding to the 50-S subunit was entirely dependent on Mg2+ and was considerably increased in the presence of tetracycline. Tris+ inhibited the coded binding while NH4+ had no pronounced effect. Even in the absence of poly A, a considerable quantity of oligolysyl-tRNA was bound to the 50-S subunits. Unlike the poly A-dependent binding, the uncoded oligolysyl-tRNA binding to the 50-S subunits was strongly inhibited by NH4+. n nThe binding of oligolysyl-tRNA to a mixture of 30-S and 50-S subunits was entirely dependent on Mg2+ and was stimulated by NH4+. The coded interaction was inhibited only slightly by Tris+ and to 50% by tetracycline. n nThe presence of initiation factors and of GTP had no effect on the interaction between oligolysyl-tRNA and the 30-S or 50-S ribosomal subunits.

Study of conditions for the inhibitory effect of N-tosyl-l-phenylalanylchloromethane on protein synthesis and the possibility of existence of different forms of the elongation factor S3 in Bacillus stearothermophilus

Abstract Exposure of the S-100 supernatant fraction from Bacillus stearothermophilus to N- tosyl- l -phenylalanylchloromethane (Tos-TheCH2Cl) destroys its ability to stimulate the poly(U)-directed polyphenylalanine synthesis in vitro. The rate of inhibition is markedly increased if free GDP or GTP are present simultaneously with the inhibitor. The inhibitory effect of Tos-PheCH2Cl in the presence of GDP seems to be highly specific because already at a ratio of 1 mole of Tos-PheCH2Cl plus 1 mole of GDP to 1 mole of S3 ★ factor (in the S-100 supernatant) the synthesizing activity of the supernatant is abolished by about 50 % (at pH 7.8). The apparent pH optimum of the inhibition is in the alkaline region with a pK at about 8.5. The position of the pK is not affected by GDP. S-100 supernatants prepared from cells harvested during the early stationary phase of growth were found to be substantially more sensitive to the combined effect of GDP plus Tos-PheCH2Cl than those prepared from the cells harvested during the logarithmic phase of growth. The possibility of the existence of two transient forms of S3 factor during the period of bacterial growth is discussed.

The role of aminoacyl-tRNA binding site on the factor EF-T in uncoupled GTPase reaction.

The properties of the N-tosyl-L-phenylalanyl chloromethane treated EF-T factor were studied in a ribosomal system in which splitting of GTP occurs. The action of N-tosyl-L-phenylalanyl chloromethane inhibits the binding of aminoacyl-tRNA to the EF-T factor. The binding and exchange of guanosine phosphates continued to be preserved. The inhibited factor is inactive in the GTPase reaction which depends on the participation of ribosomes and aminoacyl-tRNA. The uncoupled GTPase reaction (which is not dependent on the presence of aminoacyl-tRNA) is also sensitive to the effect of the inhibitor. The inhibition of the uncoupled GTPase is incomplete. These findings are attributed to the involvement of the aminoacyl-tRNA binding site of the EF-T factor in the uncoupled GTPase reaction.