THE ALLOSTERIC 3-SITE MODEL OF ELONGATION CANNOT BE CONFIRMED IN A WELL-DEFINED RIBOSOME SYSTEM FROM ESCHERICHIA-COLI

For the functional role of the ribosomal tRNA exit (E) site, two diffe rent models have been proposed, It has been suggested that transient E -site binding of the tRNA leaving the peptidyl (P) site promotes elong ation factor G (EP-G)-dependent translocation by lowering the energeti c barrier of tRNA release [Lill, R., Robertson, J. M. & Wintermeyer, W . (1989) EMBO J. 8, 3933-3938], The alternative ''allosteric three-sit e model'' [Nierhaus, K. H. (1990) Biochemistry 29, 4997-5008] features stable, codon-dependent tRNA binding to the E site and postulates a c oupling between E and aminoacyl (A) sites that regulates the tRNA bind ing affinity of the two sites in an anticooperative manner. Extending our testing of the two conflicting models, we have performed transloca tion experiments with fully active ribosomes programmed with heteropol ymeric mRNA. The results confirm that the deacylated tRNA released fro m the P site is bound to the E site in a kinetically labile fashion, a nd that the affinity of binding, i.e., the occupancy of the E site, is increased by Mg2+ Or polyamines. At conditions of high E-site occupan cy in the posttranslocation complex, filling the E site with aminoacyl -tRNA had no influence on the E site, i.e., there was no detectable an ticooperative coupling between the two sites, provided that second-rou nd translocation was avoided by removing EF-G. On the basis of these r esults, which are entirely consistent with our previous results, we co nsider the allosteric three-site model of elongation untenable, Rather , as proposed earlier, the E site-bound state of the leaving tRNA is a transient intermediate and, as such, is a mechanistic feature of the classic two-state model of the elongating ribosome.