The Effect of tRNA Derivatives Bound with Natural or Synthetic mRNA on the Interaction of Escherichia coli Ribosomes with Colicin E3

Abstract

Ribosomal binding complexes directed by poly(U) or T4 mRNA were formed with aminoacyl-tRNA or its derivatives bound to predominantly the P or A binding site. The defined binding complexes were reacted with colicin E3 and the reaction was assessed by the ability of the complexes to proceed with polypeptide synthesis. The results indicated that only one of the four complexes tested was completely resistant to colicin E3-induced inactivation: that of Phe-tRNA bound in the presence of poly(U) to the A-site. The poly(U) directed complex of AcPhe-tRNA and the T4-mRNA-directed complex at the A-site appeared slightly resistant, while the T4 mRNA initiation complex was inactivated by colicin E3 in a manner similar to non-complexed ribosomes. Colicin E3 added to ribosomes after protein synthesis had been initiated affected the subsequent polymerization in a manner corresponding to the response of the binding complexes. Thus, poly(U)-translating ribosomes were less affected than ribosomes translating the viral mRNA. The vulnerability of natural-mRNA-directed binding complexes to inactivation by colicin E3 is in accord with the mode of inactivation by the colicin in vivo.

On the interaction of colicin E3 with the ribosome

“…Similarly, tRNA and colE3 binding are mutually exclusive. These predictions are in agreement with experimental results that mRNA as well tRNA confers protection of ribosomes from colE3 attack [33,34]. In contrast, the P-site is free to bind tRNA in the presence of colE3.…”

Interaction of unfolded tRNA with the 3′-terminal region ofE. coli16S ribosomal RNA

“…The ribosomes which are engaged in the protein elongation are protected against the desactivation with the bacteriocin. It could be demonstrated by in vitro experiments that this protection can be achieved also by binding of aminoacyl-tRNA to ribosomes (25). Both enzymatic and non enzymatic binding of the aminoacyl tRNA have a similar protective effect.…”

Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes

“…Previous studies of the mechanism of colicin E3 action on the ribosome focused on the structure of ribosomes, experimental conditions required for cleavage to occur and the specific cleavage site ( Boon, 1972 ; Bowman, 1972 ; Dahlberg et al ., 1973 ; Kaufmann and Zamir, 1973 ; 1975 ; Ohno-Iwashita and Imahori, 1977 ; Jakes and Zinder, 1974b ). Very few investigations into the stage of translational arrest have been performed.…”

“…In the case of colicin E3, it is not clear whether the initiation complex can be formed once the cleavage has taken place. It was reported that colicin E3-treated ribosomes were unable to form initiation complexes ( Tai and Davis, 1974 ; Tai, 1975 ), whereas the initiation complex was formed when natural mRNA was used ( Kaufmann and Zamir, 1975 ). The effect of colicin E3 on other steps of translation was not studied.…”