Effect of nucleotide substitution on the peptidyltransferase activity of 2'(3')-O-(aminoacyl) oligonucleotides

Abstract

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.

Movement of the 3′‐end of tRNA through the peptidyl transferase centre and its inhibition by antibiotics

“…Circumstantial evidence suggests that the acceptor substrate region of the tRNA interacts there [39, 40] since, first, puromycin can bind and react with a donor substrate [41], second, both the accuracy and rate of elongation are affected in a systematic way by mutations in the 23S rRNA [42, 43] and, third, peptidyl transferase antibiotics can modulate the affinity of aminoacyl‐tRNA for the ribosome, after GTP hydrolysis and release of EF‐Tu‐GDP [44, 45]. However, interactions appear to have a low degree of site specificity [46] and, moreover, mutational studies of the ‐CCA sequence of the acceptor substrate revealed a low level of base specificity, although changes in the 3′‐terminal‐CA sequence did produce some lowering of acceptor activities in the ‘fragment’ reaction [47].…”

Functional transfer RNAs with modifications in the 3'-CCA end: differential effects on aminoacylation and polypeptide synthesis.

“…1). Chemical and nuclease protection studies indicate that the 3'-CCA sequence also functions in later steps of protein synthesis: in formation of a ternary complex between aminoacyl-tRNA, elongation factor Tu, and GTP (1) and in the binding of tRNA to 23S rRNA at the A, P, and E sites of the 50S ribosomal subunit during the elongation and translocation steps of the translation cycle (2)(3)(4)(5)(6).…”

Functional Importance of the 3′-Terminal Adenosine of tRNA in Ribosomal Translation

“…4A). Substitutions in the CCA sequence have also been shown to have only small effects on the acceptor activity of aminoacyl-oligonucleotides in the peptidyl transfer reaction (8).…”

“…The importance of the CCA end in ribosome-catalyzed peptide bond formation is well established (1,7). Chemically synthesized aminoacyl oligonucleotides were used to demonstrate the significance of the 3Ј-CCA sequence as a peptide acceptor during peptide bond formation on ribosomes (8). E. coli tRNA Val with mutations in the 3Ј-CCA sequence inhibits the peptidyltransferase activity of the ribosome (9).…”