Elongation factor EF‐P is a soluble protein that stimulates peptide bond synthesis catalyzed by the 50‐S ribosomal subunit. This factor was previously identified and characterized based on its ability to promote the synthesis of formylmethionine‐puromycin. In the present work, we tested the ability of EF‐P to promote peptide bond synthesis between ribosome‐bound fMet‐tRNA and several analogues of the 3′ terminus of aminoacyl‐tRNA, i.e. the cytidylyl(3′‐5′)‐[2′(3′)‐O‐l‐aminoacyladenosines]. EF‐P promoted synthesis to the greatest extent with certain acceptors which were otherwise inefficient in the peptidyl transferase reaction. This activity of EF‐P could not be replaced by the other soluble proteins known to be involved in polypeptide synthesis, such as EF‐Tu, EF‐Ts and EF‐G. One role of EF‐P in protein synthesis may be to allow peptide bond synthesis to occur more efficiently with some aminoacyl‐tRNAs that are poor acceptors for the ribosomal peptidyl transferase.

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.

The effect of the nature of the amino acid residue on the acceptor activity of substrates of ribosomal peptidyl transferase was investigated. We tested 2′(3′)‐O‐aminoacyladenosines containing the following amino acid residues: l‐alanine, l‐glutamine, glycine, l‐3‐(1‐benzyl‐4‐imidazolyl)‐alanine, l‐leucine, l‐lysine, l‐methionine, l‐methionine S‐oxide, l‐phenylalanine, d‐phenylalanine, l‐proline, l‐serine and l‐valine as acceptors of the acylaminoacyl residue transferred from peptidyl‐tRNA. The acceptor activity of these compounds was dependent on the nature of the side chain of the amino acid residues bound to adenosine. The acceptor activity was also affected by the nature of the donor tRNA derivative. With l‐acetylphenylalanyl‐tRNA or l‐acetylleucine npetanucleotide fragments derived from l‐acetylleucyl‐tRNA donating the acetylphenylalanyl and the acetylleucyl residue, respectively, a high acceptor activity was shown by the basic 2′(3′)‐O‐l‐lysyladenosine or 2′(3′)‐O‐l‐3‐(1‐benzyl‐4‐imidazolyl)‐alanyladenosine which acted only as weak acceptors of lysine peptides from (Lys)n‐tRNA. The transfer reaction catalyzed by peptidyl transferase was stereospecific with respect to acceptor substrates. The acylaminoacyl residue was transferred from tRNA to 2′(3′)‐O‐l‐phenylalanyladenosine, whereas 2′(3′)‐O‐d‐phenylalanyladenosine, containing a d‐phenylalanine residue, was completely inactive as acceptor.

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In the course of protein biosynthesis, the 3'-ends of aminoacyl-tRNA (aa-tRNA) and peptidyl-t RNA specifically interact with macromolecules of the protein biosynthesis machinery. The 3'-end of tRNA consists of an invariant C-C-A single strand. Interaction of the aminoacyl-tRNA 3'-end with elongation factor Tu (EF-Tu) containing bound GTP is necessary for the formation of the aa-tRNA. EF-TU . GTP complex and, after the complex binds to the ribosome, for the GTP hydrolysis. This process is followed by the specific binding of the aminoacyl-tRNA 3'-end to the aminoacyl (A) site of the ribosome. In this review, a model is proposed that involves Watson-Crick base pairing of the C-C sequence of the aminoacyltRNA 3'-end with a specific G-G sequence of the ribosomal 23s RNA. Similarly, peptidyltRNA binds with its 3'-end to the peptidyl (P) site of the ribosome. This binding may also involve Watson-Crick base pairing of the C-C-A sequence with a complementary sequence of 23s RNA. It is proposed that peptide bond formation is catalyzed by a functional site of the 23s RNA located near the 3'-ends of aminoacyl-tRNA and peptidyl-tRNA. A model is suggested in which two loops of the 23s RNA, brought into close proximity via folding, are involved both in binding the 3'-ends of the tRNAs and in catalyzing peptide bond formation. This model presumes a dynamic structure for ribosomal RNA, which is modulated by interaction with elongation factors and ribosomal proteins.