THE 2 MAIN STATES OF THE ELONGATING RIBOSOME AND THE ROLE OF THE ALPHA-SARCIN STEM-LOOP STRUCTURE OF 23S RNA

According to the allosteric three-site model of the elongation cycle the ribosome oscillates between two main-functional states, viz the pre-translocational state with occupied A and P sites (E site with low affinity) and the post-translocational state with occupied P and E sites (A site with low affinity). This proposition could be confirmed by a determination of the thermodynamic parameters. High activation-energy barriers were found between both states, namely about 90 kJ mol-1 at 15 MM Mg2+ for either transition (post --> pre transition = A-site binding and pre --> post transition = translocation). The various A-site states (binding of ternary complex, EF-Tu dependent GTP cleavage, peptide-bond formation) are not separated by significant activation-energy barriers. The rate-limiting step of the elongation cycle is A-site binding, and not translocation as assumed previously. The principal role of both elongation factors is the reduction of the respective activation-energy barrier, thus accelerating the rate of the elongation cycle by several orders of magnitude. Cleavage of a single phosphodiester bond after G2661 of 23S rRNA by the RNase alpha-sarcin abolishes the functions of both elongation factors on the ribosome. This observation implies that the alpha-sarcin stem-loop structure plays an important role in the ribosomal conformational changes involved in the allosteric transitions. Indeed we could demonstrate that suitable oligodeoxynucleotide probes complementary to the alpha-sarcin region induce a conformational change in the 50S subunits; this conformational change causes an irreversible dissociation of tightly coupled ribosomes upon sucrose-gradient centrifugation. The 50S remained in its inactive state even after removal of the hybridized DNA probe. The results suggest that the alpha-sarcin region plays a pivotal role in lowering the activation barriers of the conformational transitions between the post- and pre-translocational states. However, the presence of an elongation factor is essential for establishing a defined pre- or post-translocational state.