Substrate Specificity of Ribosomal Peptidyl Transferase

Abstract

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.

Peptide Bond Formation Stimulated by Protein Synthesis Factor EF‐P Depends on the Aminoacyl Moiety of the Acceptor

“…Since the puromycin reaction represents a special case insofar as acceptors for the ribosomal peptidyl transferase are concerned, we asked if EF-P had any effect on the peptidyl transferase reaction with other acceptors; and if so, whether the effect was dependent upon the nature of their aminoacyl moieties. To answer this question with a simple system which allows the examination of peptide bond synthesis apart from the other steps of translation, we used a set of aminoacyl acceptors similar to those described by Rychlik et al [4]. These workers measured the ability of 2'(3')-0-~-aminoacyladenosines to act as substrates in the peptidyl transferase reaction and found a wide variation in acceptor activities which was dependent upon the aminoacyl moiety.…”

“…However, a number of observations suggest that these assumptions should be more carefully examined. For example, (a) not all aminoacyl-tRNAs are translated with the same efficiency when programmed with synthetic templates in the presence of purified EF-T and EF-G [3]; (b) the activity of an aminoacyl acceptor in the peptidyl transferase reaction is markedly dependent upon the nature of the aminoacyl moiety [4]; (c) aminoacyl-oligonucleotides, which represent the terminal portion of aminoacyl-tRNAs, exhibit a binding affinity to the ribosomal A site that parallels their aminoacyl acceptor activity …”

Inhibition of Chloramphenicol Binding to Escherichia coli 70‐S Ribosomes by 2′(3′)‐O‐Aminoacyl‐dinucleoside Phosphates Derived from the Aminoacyl‐tRNA Acceptor Terminus

“…Thus, it follows that the high affinity constant of C-d2'A-Phe (IIIa) is responsible for the efficient interaction of this compound with the peptidyltransferase center. High inhibitory activity of puromycin derivatives of aromatic amino acids in polypeptide synthesis or more specifically the high acceptor activity of 2'(3')-O-aminoacyladenosines with aromatic amino acids have been already noted [22,23]. As was first suggested by Rychlik et LIZ.…”

“…As was first suggested by Rychlik et LIZ. [23], the side chain of the aminoacyl residue probably interacts with a specific locus at the ribosome during binding of an acceptor substrate to the peptidyltransferase A-site. This interaction appears to be strongest for the amino acids with hydrophobic side chains and weakest for glycine.…”

Puromycin–rRNA interaction sites at the peptidyl transferase center

“…The effects at A508 and A1579 may be because of (1) an allosteric effect transmitted along the peptide channel; (2) the presence of a secondary puromycin binding site that may or may not have functional relevance; or (3) peptidyl-puromycin release along the peptide channel+ We favor explanation (1), the allosteric effect, for the following reasons+ First, any binding of puromycin to a secondary ribosomal site is very weak and would probably not yield a footprint (Bourd et al+, 1983)+ Second, although both nucleotides lie close to the peptide exit site (Müller et al+, 2000), as a major effort was made to prepare 70S ribosomes and 50S subunits that were free of peptidyl-tRNA (Mahkno et al+, 1988), the identities and relative yields of all the nucleotide protections and enhancements were unaltered for E. coli 70S ribosomes, 50S subunits, and polysomes+ This renders it very unlikely that the A508 and A1579 effects are the result of puromycin-mediated peptide release along the peptide channel in our experiments+ However, such a process could explain the earlier puzzling result, obtained in more crudely prepared ribosomes, of a direct crosslink of [ 3 H]-p-azidopuromycin to L23 (Nicholson et al+, 1982a), which directly borders A508 and A1579 at the peptide-exit site (Fig+ 6B)+ Puromycin perturbs the position of the 39 end of a deacylated tRNA substrate bound in the P-site, leading to strong reductions in the tRNA crosslinks to F29 and F49+ Other peptidyl-transferase antibiotics also affect the yields of crosslinking, but only chloramphenicol and pristinamycin IIA produce such dramatic, albeit differing, effects (Kirillov et al+, 1999)+ Pristinamycin IIA abolished the F29 and F49 crosslinks and reduced the crosslink to F19, whereas chloramphenicol selectively abolished F29+ Therefore, most of the peptidyl-transferase drugs, including those previously assigned to the A-site, directly affect the positioning of the 39 end of P9-site-bound tRNA+ Because puromycin is considered to be a structural analog of the 39 end of an aminoacyl-tRNA substrate, it is reasonable to expect that there might be some correlation between the chemical footprints of puromycin and that of a tRNA substrate bound in the ribosomal A site+ Removal of the 39-terminal adenosine from yeast tRNA Phe , although not from E. coli tRNA Phe , bound in the presence of P-site-bound deacylated tRNA, resulted in increased modification of G2553 on E. coli ribosomes (unpubl+ data cited in Moazed & Noller, 1989)+ Moreover, removal of the acyl moiety led to increased reactivities of ⌿2555, A2602, and U2609 in E. coli 23S rRNA (Moazed & Noller, 1989)+ Although G2553 is protected by puromycin, none of the other nucleotides are affected+ This may reflect the fact that puromycin includes adenosine and 39-linked aminoacyl moieties (Fig+ 1), rather than a tRNA population containing a mixture of 29-and 39-linked aminoacyl groups in rapid equilibration via acyl transfer (Symons et al+, 1978;Moazed & Noller, 1989)+ Little is known about the molecular specificity of puromycin interactions with the ribosome+ It is unlikely to be from base pairing because the analogs, 1-N 6 -ethenoadenosine-Phe (Chladek et al+, 1976), inosine-L-Phe, cytidine-L-Phe, and 3-N 4 -ethenocytidine-Phe (Rychlik et al+, 1970)…”