Peptide bond formation in protein biosynthesis takes place by transfer of the growing peptidyl group from the CCA terminus of 1 molecule of transfer RNA (tRNA) to an aminoacyl group attached to the CCA terminus of a second, incoming, molecule of tRNA.1-4 There is increasing evidence that the reaction is catalyzed by an enzyme, peptidyl transferase, which is an integral part of the 50S ribosomal subunit.5-7 The present communication is concerned with substrate specificity at this catalytic center.The peptidyl transfer reaction is normally linked to other reactions of protein biosynthesis through simultaneous interactions of the tRNA molecules with template, various parts of the ribosome, and, possibly, soluble protein factors (ref. 5: review). By use of substrate analogs which lack certain functional parts of the tRNA molecule but retain others, it is possible to resolve peptidyl transfer from the other reactions. Aminoacyl-tRNA can be replaced by puromycin (an analog of aminoacyl-adenosine) and peptidyl-tRNA by CAACCAMet-F (a fragment from F-Met-tRNA). The puromycin reaction has been extensively studied and has provided much information on protein biosynthesis.5 8 The development of a system in which CAACCA-Met-F acts as a peptidyl donor (with puromycin as acceptor) is a recent development.9The reaction of CAACCA-Met-F with puromycin to give N'-formyl-methionylpuromycin is catalyzed by washed 50S ribosomal subunits6 and is dependent upon the presence of monovalent cations, divalent cations, and alcohol,9 10 whereas 30S subunits, soluble protein factors, and guanosine 5'-triphosphate are not required.6 Evidence that the fragment reaction takes place by the same mechanism as peptidyl transfer in protein biosynthesis is provided not only by the involvement of common factors but also by the observation that certain antibiotic inhibitors of protein biosynthesis are very active in the system.11It is widely believed that there are two substrate binding sites on ribosomes,5, 12-16 one (the P-site) for holding the peptidyl-tRNA, the other (the A-site) for attachment of the aminoacyl-tRNA. Replacement of the normal substrates of protein biosynthesis by fragments obtained from them, and other small analogs such as puromycin, provides a means to investigate interactions which occur at the two sites in the neighborhood of the catalytic center. Studies with analogs of aminoacyl-tRNA have already provided information on specificity at the A-site.171-9 In the present work we have investigated the activities of various analogs of peptidyl-tRNA in order to study the specificity at the P-site. Structural features considered are size and base sequence of the oligonucleotide, acylation of the a-amino group, and nature of the aminoacyl residue.Materials and Methods.-Salt-washed ribosomes, ribosomal subunits, and postribosomal supernatant fraction (S-100) were prepared from E. coli MRE 600 by procedures described elsewhere.7 10 Sources of standard materials are noted in the same communica-1042
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.
The effect of antibiotics on the coded binding of peptidyl-tRNA to the ribosome and the transfer of the peptidyl residue from peptidyl-tRNA to puromycin has been studied. The coded binding of polylysyl-tRNA was inhibited only by tetracyclines, the binding of acetylphenylalanyl-tRNA was inhibited by tetracyclines and streptomycin and stimulated by spiramycin and carbomycin. The most potent inhibitors of the puromycin reaction with polylysyl-tRNA were the macrolides erythromycin, spiramycin, carbomycin and oleandomycin, inhibiting by 50°/, at a concentration of 0.1 pM; neomycin (1 pM), chloramphenicol (100 yM), tetracyclines (100 pM), streptomycin (50 pM) and streptovitacin A (1 mM) were somewhat less effective. The puromycin reaction with acetylphenylalanyl-tRNA was inhibited by antibiotics in a similar way, only erythromycin stimulated this reaction a t concentrations between 0.1 and 500 pM.Since Sorm and Grunberger [I] and Gale and Folkes [Z] demonstrated that the biological effect of chloramphenicol consists in an inhibition of protein biosynthesis, other antibiotics with a similar effect have been discovered and much effort has been expended to elucidate the mechanisms by which these substances inhibit protein synthesis. Recently, the cell-free systems capable of protein synthesis in vitro have been simplified to such an extent that it has become possible to investigate the effect of antibiotics not only on the overall process of de novo protein biosynthesis [3-71 but also on particular stages of this process [8--12]. Most attention has been paid to the effect of antibiotics on binding of aminoaoyltRNA to ribosomes [7,13-171. In this paper we shall describe the effect of some antibiotics on the ribosomal stages of protein biosynthesis : formation of the ternary complex between polypeptidyl-tRNA ribosomes and mRNA, and formation of peptide bond on ribosomes represented in its simplified form by the puromycin reaction, i. e.by transfer of the nascent polypeptide from polypeptidyl-tRNA to puromycin. A part of these results has been published in preliminary form [18,19]. We did not find any difference between the activities of both preparations under our experimental conditions.The interaction of [14C]polylysyl-tRNA and AcPhe-tRNA with ribosomes [19] and the release of lysine peptides and of the AcPhe residue by puromycin [18] was determined as described earlier.Poly A and Poly U were obtained from Calbiochem (U.S.A.). D-threo-Chloramphenicol, L-threo-chlor-
Digestion of ribosomes by ribonuclease T, results in the inactivation of peptidyl transferase and of the binding ability of the donor site. However, the binding activity of the acceptor site is not affected by ribonuclease digestion. The binding sites of ribonuclease-treated ribosomes retain their original sensitivity to chloramphenicol, spiramycin, erythromycin and lincomycin.Digestion of 50-5 ribosomal subunits by ribonuclease T, results in the loss of peptidyl transferase activity and of the binding ability of the donor site in the same way as digestion of 70-5 ribosomes either intact or recombined from 30-5 and 50-S subunits. The acceptor activity of the acceptor binding site of 50-S ribosomal subunits does not change after T, ribonuclease digestion. Erythromycin, bound to the ribosomes before digestion by ribonuclease TI, decreases the rate of inactivation of peptidyl transferase, whereas spiramycin does not change the effect of ribonuclease on transfer activity.
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