Stanislav Chládek

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.

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 peptidyltransferase center of Escherichia coli ribosomes: Binding sites for the cytidine 3′-phosphate residues of the aminoacyl-tRNA 3′-terminus and the interrelationships between the acceptor and donor sites

The substrate specificity of the acceptor site of peptidyltransferase of Escherichia coli 70 S ribosomes was investigated in Ac-Phe-tRNA . poly(U) . 70 S ribosome (system A) and tRNC-A-Phe . poly(U) . C-A-C-C-A-Phe . 70 S ribosome (system B) systems by using C-C-A-Gly, C-C-A-Phe, C-A-Gly and C-A-Phe as analogs of the 3-terminus of aminoacyl-tRNA. It was found that an addition of CP residue to C-A-Gly and C-APhe resulted in an increase of the acceptor activity in system A; the increase is more remarkable for C-A-Gly than for C-A-Phe, while the acceptor activities of C-C-A-Gly and C-C-A-Phe are roughly similar. On the other hand, dramatically increased binding affinities of C-C-A-Phe and C-C-A-Gly relative to C-A-Phe and C-A-Gly for the A site of peptidyltransferase were observed in system B using an inhibition assay; C-C-A-Phe binds much more strongly than C-C-A-Gly. The results indicate the important role of the third CP residue and the aminoacyl moiety of the 3-terminus of aminoacyl-tRNA in the interaction with the acceptor site of peptidyltransferase, as well as the existence of cooperative effects between A and P sites of peptidyltransferase. These effects, depending on an occupancy of P site, may significant the specificity of the peptidyltransferase A site.

Inhibition of the peptidyltransferase acceptor site by 2′(3′)-O-cycloleucyl- and α-aminoisobutyryl derivatives of cytidylyl-(3′–5′)adenosine☆

2(3)-O-(N-Benzyloxycarbonylcycloleucyl)adenosine (1a) was prepared by esterification of 5-O-(4-methoxytrityl)adenosine with N-benzyloxycarbonylcycloleucine in the presence of dicyclohexylcarbodiimide and subsequent deprotection in acidic medium. The compound 1a was separated into pure 2- and 3-isomers using HPLC; these isomers were found to undergo an easy interconversion. Compound 1a was coupled with N-dimethylaminomethylene-2,5-di-O-tetrahydropyranylcytidine 3-phosphate in the presence of dicyclohexylcarbodiimide to give, after subsequent deblocking, cytidylyl(3 leads to 5)2(3)-O-cycloleucyladenosine (1c). Compound 1c, as well as the related cytidylyl(3 leads to 5)2(3)-O-(alpha-aminoisobutyryl)adenosine (1d), inhibited the peptidyltransferase catalyzed transfer of an AcPhe residue to puromycin in the Ac[14C]Phe-tRNA . poly(U) . 70 S E. coli ribosome system. A half of the maximum inhibition of AcPhe-puromycin formation (at 10(-5) M puromycin) was achieved at 9.5 . 10(-6) M of compound 1c and 9 . 10(-5) M of compound 1d, respectively. The inhibition of the puromycin reaction by compound 1d shows a mixed-type of inhibition kinetics. Further, none of the compounds 1c and 1d was an acceptor in the peptidyltransferase reaction. Both compounds 1c and 1d inhibited the binding of C-A-C-C-A[14C]Phe to the A site of peptidyltransferase in a system containing tRNAPhe . poly(U) . 70 S E. coli ribosomes, in which compound 1d was a much stronger inhibitor than 1c. These results indicate that the derivatives such as compounds 1c and 1d which contain an anomalous amino acid with a substituent in lieu of alpha-hydrogen can interfere with the peptidyltransferase A site; however, they are not acceptors in the peptidyltransferase reaction probably due to a misfit of the alpha-substituent.

Effect of thiostrepton and 3′-terminal fragments of aminoacyl-tRNA on EF-Tu and ribosome-dependent GTP hydrolysis

The effect of the antibiotics thiostrepton and micrococcin on EF-Tu-catalyzed (ribosome-dependent) GTP hydrolysis in the presence of A-Phe, C-A-Phe, or C-C-A-Phe (related to the sequence of the 3-terminus of aminoacyl-tRNA)(System I) or by methanol (uncoupled GTPase, System II) was investigated. In System I, thiostrepton increases the binding affinities of the effectors to the EF-Tu.GTP.70 S ribosome complex, as well as the extent of the GTP hydrolysis, while the KmGTP is virtually unchanged. Similarly, in the uncoupled system (System II) and in the absence of effectors, thiostrepton significantly increases VmaxGTP, whereas KmGTP remains unaffected. Micrococcin is without any effect in both systems. The uncoupled GTPase (in System II) is also strongly inhibited by C-A-Phe. The results indicate the crucial role of the EF-Tu site which binds the aminoacylated C-C-A terminus of aminoacyl-tRNA in promoting GTP hydrolysis. It follows that the binding of the model effectors (such as C-C-A-Phe) to that site is favorably influenced by the interaction of thiostrepton with the 50 S ribosomal subunit, whereas thiostrepton, per se, does not influence the affinity of EF-Tu for GTP.

The Synthesis, Reactions, and Properties of the 2′(3′)-O-Aminoacyl and Peptidyl Nucleosides and Nucleotides

The main impetus for the studies of the chemistry of the title compounds stems from the fact that the compounds from the aminoacyl transfer ribonucleic acid (aa-tRNA; see Section 11 for abbreviations used in this chapter), the key intermediate in the biosynthesis of proteins. In 1957 it was discovered that, during intermediate steps of protein synthesis, amino acids are bound to tRNA.(1) It was subsequently determined that the amino acids are linked by an ester bond to the 3′-terminal adenosine of the ubiquitous C-C-A sequence of tRNA(2-4) (Scheme 1). This was clearly indicated by evidence that 2′(3′)-0-leucyladenosine was produced by the ribonuclease digestion of Leu-tRNA.(2) Puromycin (2), a naturally occurring antibiotic produced by Streptomyces alboniger, is structurally similar to the 3′-0-aminoacyl terminus of aa-tRNA, with the exception that an aminoacyl amido bond replaces the aminoacyl ester bond.

Conformational activation of the EF-Tu·GTPase activity Stimulation of the 2′(3′)-O-l-phenylalanyladenosine-promoted EF-Tu·GTPase upon binding of the tRNAPhe lacking the terminal adenosine residue

The elongation factor Tu (EF-Tu) dependent GTPase (in the presence of aurodox) is stimulated by analogs of the aminoacyl tRNA 3′-terminus in the following order: A-Phe < C-A-Phe < C-C-A-Phe. The GTPase-promoting activity of A-Phe is strongly enhanced by tRNA-C-C (devoid of 3′-terminal adenosine residue) but not by intact tRNA-C-C-A. On the other hand, the activity of C-A-Phe as the EF-Tu·GTPase promoter is only slightly enhanced by tRNA-C-C.

The elongation factor Tu.GTPase reaction: effect of 2'(3')-O-aminoacyl oligoribonucleotides.

The activity of synthetic (2(3)-O-aminoacyl trinucleotides, C-C-A-Phe, C-C-U-Phe, C-U-A-Phe, U-C-A-Phe and C-A-A-Phe, in promoting the EF-Tu.70 S ribosome-catalyzed GTP hydrolysis was investigated. It was found that the activity decreases in the order C-C-A-Phe greater than C-U-A-Phe greater than U-C-A-Phe greater than C-A-A-Phe much greater than C-C-U-Phe. Thus, the substitution in natural C-C-A sequence with other nucleobases weakens binding of 2(3)-O-aminoacyl trinucleotides to EF-Tu, with the substitution at the 3-position having the most profound effect. Since the 2(3)-O-aminoacyl oligonucleotides mimic the effect of the aa-tRNA 3-terminus on EF-Tu.GTPase, it follows that EF-Tu probably directly recognizes structure of nucleobases in the aa-tRNA 3-terminus, with the 3-terminal adenine playing the most important role.

Aminoacyl derivatives of nucleosides, nucleotides, and polynucleotides: XVI. Effect of some anions on the reaction course of petptide synthesis with active esters

Abstract The reaction of 2′,3′-O-aminomethylethoxymethyleneadenosine (I) with p-nitrophenyl and N-hydroxysuccinimide esters of α, e-dibenzyloxycarbonyl- l -lysine (IIa and IIb) and N- benzyloxycarbonyl- l -valine p-nitrophenyl ester (IIc) in the presence of formate or acetate ions is described. In addition to dipeptide orthoesters (IIIa, IIIb) N-formyl or N-acetyl derivatives (IIIc, IIId) were obtained in amounts depending on the type of anion and active ester used. A similar “anion exchange” reaction has also been observed under the conditions currently used for peptidation of aminoacyl-tRNAs.