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Seven 2'(3')-O-(aminoacyl) trinucleotides with structures derived from the 3'-terminal C-C-A sequence of aa-tRNA via nucleotide substitutions were investigated as acceptor substrates in the peptidyltransferase reaction and as inhibitors of substrate binding to the peptidyltransferase A site. It was found that all tested compounds were active in both systems, although substitution in the first and second nucleotide position results in some decrease of acceptor activity. Remarkably, replacement of natural cytidylic acid residues in C-C-A-Phe with guanylic acid moieties resulted only in a small decrease of acceptor or binding activity. The results indicate that the acceptor sequence of aa-tRNA is not probably engaged in base pairing with a sequence of 23S RNA during its interaction with the peptidyltransferase A site.

Wahrend der Proteinbiosynthese wechselwirken das 3′-Ende der Aminoacyl-tRNA (aatRNA) und der Peptidyl-tRNA spezifisch mit Makromolekulen des Proteinbiosynthese-Apparats. Das 3′-Ende der tRNAs besteht aus einem invarianten C-C-A-Einzelstrang. Die Wechselwirkung des 3′-Endes der aa-tRNA mit dem Elongationsfaktor (EF) ist wichtig fur die Bildung des aa-tRNA·EF-Tu·GTP-Komplexes und, nachdem dieser Komplex an das Ribosom gebunden ist, fur die GTP-Hydrolyse. Diesem Vorgang folgt die spezifische Bindung des 3′-Endes der Aminoacyl-tRNA an die Acceptorstelle der ribosomalen Peptidyltransferase. In diesem Aufsatz wird ein Modell vorgestellt, nach welchem die C-C-Nucleotide des 3′-Endes der Aminoacyl-tRNA mit einer spezifischen G-G-Sequenz der ribosomalen 23S-RNA Watson-Crick-Basenpaare bilden. Ahnlich bindet die Peptidyl-tRNA mit ihrem 3′-Ende an die komplementare Sequenz der ribosomalen 23S-RNA. Wir schlagen vor, das die Bildung der Peptidbindung zwischen den beiden tRNAs durch einen Bereich der 23S-RNA katalysiert wird, der sich in der Nahe des 3′-Endes der Aminoacyl- und der Peptidyl-tRNA befindet. An der Bindung der 3′-Enden der beiden tRNAs sowie an der Katalyse sind zwei Schleifen der 23S-RNA beteiligt, welche durch Faltung in unmittelbare Nachbarschaft gebracht werden konnen. Das vorgeschlagene Modell setzt eine dynamische Struktur der ribosomalen RNA voraus, die durch Wechselwirkungen mit Elongationsfaktoren und ribosomalen Proteinen funktionell verandert und gesteuert wird.

The specific synthesis of the 2′(3′)-0-aminoacyl triribonucleoside diphosphates, analogs of the 3′-terminus of AA-tRNA is described via phosphotriester intermediates.

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 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 tRNAPhe·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-A-Phe 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 significantly influence the specificity of the peptidyltransferase A site.

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.

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.

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-tRNA's.

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.

Wahrend der Proteinbiosynthese wechselwirken das 3′-Ende der Aminoacyl-tRNA (aatRNA) und der Peptidyl-tRNA spezifisch mit Makromolekulen des Proteinbiosynthese-Apparats. Das 3′-Ende der tRNAs besteht aus einem invarianten C-C-A-Einzelstrang. Die Wechselwirkung des 3′-Endes der aa-tRNA mit dem Elongationsfaktor (EF) ist wichtig fur die Bildung des aa-tRNA·EF-Tu·GTP-Komplexes und, nachdem dieser Komplex an das Ribosom gebunden ist, fur die GTP-Hydrolyse. Diesem Vorgang folgt die spezifische Bindung des 3′-Endes der Aminoacyl-tRNA an die Acceptorstelle der ribosomalen Peptidyltransferase. In diesem Aufsatz wird ein Modell vorgestellt, nach welchem die C-C-Nucleotide des 3′-Endes der Aminoacyl-tRNA mit einer spezifischen G-G-Sequenz der ribosomalen 23S-RNA Watson-Crick-Basenpaare bilden. Ahnlich bindet die Peptidyl-tRNA mit ihrem 3′-Ende an die komplementare Sequenz der ribosomalen 23S-RNA. Wir schlagen vor, das die Bildung der Peptidbindung zwischen den beiden tRNAs durch einen Bereich der 23S-RNA katalysiert wird, der sich in der Nahe des 3′-Endes der Aminoacyl- und der Peptidyl-tRNA befindet. An der Bindung der 3′-Enden der beiden tRNAs sowie an der Katalyse sind zwei Schleifen der 23S-RNA beteiligt, welche durch Faltung in unmittelbare Nachbarschaft gebracht werden konnen. Das vorgeschlagene Modell setzt eine dynamische Struktur der ribosomalen RNA voraus, die durch Wechselwirkungen mit Elongationsfaktoren und ribosomalen Proteinen funktionell verandert und gesteuert wird.

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.