The substrate specificity of the acceptor site of peptidyltransferase of Escherichia coli 70S ribosomes was investigated in the fMet-tRNA.A-U-G.70S ribosome and AcPhe-tRNA.poly(U).70S ribosome systems by using a series of 2'- and 3'-aminoacyldinucleoside phosphates as acceptors. These chemically synthesized compounds are analogues of the 3' termini of either 2'(3')-, 2'-, or 3'-aminoacyl transfer ribonucleic acids (AA-tRNAs) of the types C-A-aa, C-2'-dA-aa, C-3'-dA-aa, C-3'-dA-3'-NH-aa, and C-2'-dA-2'-NH-aa (aa = Phe, D-Phe, Lys, Leu, Ala, Glu, Pro, Gly, Asp, Met, and alpha-aminoisobutyryl). It was found that the 3'-aminoacyl derivatives of optically active amino acids are much better acceptors of N-formyl-L-methionine (fMet) or N-acetyl-L-phenylalanine (AcPhe) residues than the isomeric 2'-aminoacyl derivatives with affinity constant ratios (KM 2'/3') greater than 100. Likewise, C-A(D-Phe) is a weaker acceptor than the corresponding L derivative C-A-Phe. In contrast, all glycyl derivatives (C-2'-dA-Gly, C-3'-dA-Gly, C-3'-dA-3'-NH-Gly and C-2'-dA-2'-NH-Gly) are good acceptors of the fMet residue, with ratios (KM 2'/3') of approximately 2. On the basis of these results, a model for the stereochemical control of the peptidyl-transferase reaction is proposed. It assigns a major role to the orientation of the amino acid side chain in 2'- or 3'-AA-tRNA. A detailed model of the interaction of the acceptor terminus of 3'-AA-tRNA with the acceptor site of peptidyltransferase is also proposed. The model is strikingly similar to those for the active sites of proteolytic enzymes.

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The chemical synthesis of C-A(2'Me)Phe (XIII), C-A(2'Phe)Me (Xlla), C-A(2'Phe)H (Xllb), and C-A-Phe (XV) as well as the precursory nucleoside derivatives, A-(2'Me)Phe (Vb), A(2'Phe)Me (Va), and A(2'Phe)H (IX), is described. "Nonisomerizable analogs" of the 3' terminus of AA-tRNA compounds, XIII, Xlla, Xllb, Va, Vb, and IX, were tested as acceptor substrates of ribosomal peptidyltransferase. The 3'-0-aminoacyl derivatives C-A(2'Me)Phe T A he exact location of the aminoacyl residue on the terminal adenosine unit of AA-tRNA* 1 has been a matter of considerable interest (Zachau and Feldman, 1965). But, because of the extremely rapid 2' +±V acyl migration within the cis vicinal diol of the terminal adenosine residue (with a half-time estimated at ca. 2 X 10™4 sec, Griffin et al., 1966), a definitive assignment of the position of the aminoacyl group in native AA-tRNA cannot be made using conventional chemical and physical methods. Furthermore, in an enzymemediated system such as protein biosynthesis, it is probable that a specific isomer of AA-tRNA is used in each step, although the same isomer may not be used throughout all of the stages; indeed, this now appears to be the case. On the basis of the structure and activity of the antibiotic puromycin, which can be considered a simple 3 '-O-aminoacyl-tRNA analog, and the inactivity of its 2' analog in the inhibition of polypeptide synthesis (Nathans and Neidle, 1963), it has been assumed that 3'-O-aminoacyl-tRNA is an active species in protein biosynthesis. Moreover, we have recently observed (Chládek et al., 1973) that the 3'-phenylalanyl ester of "opent From the Michigan Cancer Foundation, Detroit, Michigan 48201.

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A new type of nucleoside poly (5'-phosphate) analogue, nucleoside 5"-phosphoazidate, with an azido group on the terminal phosphate of GTP, ATP, GDP, GMP, AND AMP, has been synthesized by nucleophilic displacement of the corresponding activated nucleosides by azide ion in yields of 25-45%. Guanosine 5-phosphoazidates is readily photolyzed by ultraviolet light; the corresponding adenosine derivative photolyzes more slowly. Guanosine 5'-O-(2-azidodiphosphate) and guanosine 5"-O-(3-azidotrophosphate) are competitive inhibitors of the formation of the ribosome-EF-G-GDP-fusidic acid complex and of the ribosome-EF-G GTPase. The dissociation constants of the former reaction are calculated to be 27 and 7 micrometer, respectively, or 270 and 73 times that of GDP. In the latter reaction, which is conducted in the absence of fusidic acid, the Ki values are 330 and 28 micrometer, respectively, or 12 and 1 times that of GDP. Guanosine 5"-O-(2-azidodiphosphate) and guanosine 5'-O-(3-azidotriphosphate) also complete with GTP in the formation of the binary complex. EF-Tu-GTP, with respective Kd values of 750 and 75 relative to GTP.