17] Experimental prerequisites for determination of tRNA binding to ribosomes from Escherichia coli

Abstract

Publisher Summary This chapter discusses the experimental prerequisites for the determination of transfer RNA (tRNA) binding to ribosomes from Escherichia coli. In studies, the tRNA binding features of 70S ribosomes from E. coli suggests that these ribosomes contain three tRNA binding sites: the A site, which accepts the aminoacyl-tRNA during the first step of the elongation phase in protein biosynthesis, can also accept peptidyl-tRNA; the P site, where the three possible forms of a tRNA, peptidyl-, aminoacyl-, or deacyl-tRNA can bind; and the E site that shows a strong specificity for deacylated tRNA. The functional links between the three sites have been also established for the elongation phase of protein biosynthesis. The growing peptide chain is prolonged by one amino acid via three basic reactions. The first reaction is the occupation of the A site by an aminoacyl-tRNA that separates into a selection step and a tight-binding step. The selection process analyzes mainly the correctness of codon–anticodon interaction. After occupation of the A site, peptide-bond formation occurs in the second reaction: the peptidyl moiety attached to the P-site-bound tRNA is transferred to the free amino group of the aminoacyl-tRNA at the A site via a peptide bond. This reaction is catalyzed by the peptidyltransferase center located on the large subunit of the ribosome. The third reaction is the elongation factor–G·guanosine triphosphate–dependent translocation of the new peptidyl-tRNA from the A to the P site.