Are the Current Three-Site Models Valid Descriptions of the Ribosomal Elongation Cycle?

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 94; no. 20; pp. 10499 - 10500
• Main Authors: Nierhaus, Knud H., Junemann, Ralf, Christian M. T. Spahn
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 30.09.1997; National Acad Sciences; The National Academy of Sciences of the USA
• Subjects: Allosteric Regulation; Commentary; Escherichia coli; Models, Chemical; Peptide Chain Elongation, Translational; Polyribosomes; Ribosomes; Ribosomes - chemistry; RNA, Ribosomal - chemistry; RNA, Transfer - chemistry; Transfer RNA
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.94.20.10499

In the early eighties a third tRNA binding site, the E site, was found in addition to the classical A and P sites; the E site seems to be a universal feature of ribosomes. Two models exist that describe different aspects of the three sites. The hybrid-site model is based on protection experiments introducing hybrid states of tRNA binding: The tRNAs move alternately on one or the other subunits through the tRNA binding sites in the course of elongation. An analysis of the functional interplay of the three ribosomal sites led to the second model, the allosteric three-site model. The key features are that the deacylated tRNA is stably bound to the E site in a codon-dependent manner and that the A site and the E site are reciprocally linked. Both models highlight different features of the elongation cycle and do not necessarily exclude each other. Recently, the demise of the allosteric three-site model was proclaimed by attempting to demonstrate that (i) the tRNA binds to the E site in a labile fashion and (ii) no reciprocal linkage exists between the A and E sites. Concerning the first statement, the old finding was reproduced that the stability of E-site binding depends on the buffer conditions: A tRNA is stably bound to the E site and is not released upon translocation in our polyamine /Mg super(2+) buffer, in contrast to the buffer system without polyamines and low Mg super(2+). Therefore, the argument concerning labile E-site binding can be reduced to the question of which buffer conditions might reflect the in vivo situation. A thorough analysis of native polysomes derived from Escherichia coli cells demonstrated that at least 75% of the posttranslocational (POST) ribosomes carry a deacylated tRNA confirming stable binding to the E site in vivo. E site-specific protection of 23S rRNA is found in polysomes arguing in favor of an occupied E site. Also Wintermeyer and colleagues reported an occupied E site in native disomes after an incubation with EF-G for 10 min at 37 degree C. Furthermore, codon-anticodon interaction in the E site before and after a (-1) frameshift at a so-called slippery sequence of seven nucleotides requires stable E site binding in vivo. Altogether, stable binding of deacylated tRNA to the E site is a valid feature in vivo. The polyamine/Mg super(2+) buffer matches the corresponding ion concentrations in vivo.