A heuristic schematic of the complex free-energy landscape of the elongation cycle, including macrostate (MS)-I and MS-II of the pretranslocational (PRE) ribosomal complex. Conformational changes of the ribosomal complex can occur along either the reaction coordinate or the conformational space axes. Conformational changes that take place along the reaction coordinate axis correspond to the rearrangements of the ribosomal complex that facilitate the elongation reaction that will ultimately transform posttranslocation (POST)-1 into POST-2. Conformational changes along the conformational space axis, by contrast, correspond to fluctuations among the ensemble of conformers that exist at all points along the reaction coordinate, leading to the availability of numerous parallel reaction pathways, which are the hallmark of a complex free-energy landscape. The energetic barriers separating POST-1 from the MS-I state of the PRE complex and the MS-II state of the PRE complex from POST-2 are large enough such that overcoming these barriers generally requires the energy released from GTP hydrolysis by elongation factor Tu and/or peptidyl transfer (for POST-1 → MS-I transitions) and GTP hydrolysis by EF-G (for MS-II → POST-2 transitions). The energetic barrier separating MS-I from MS-II, however, is small enough such that stochastic, thermally driven fluctuations between MS-I and MS-II are permitted. In addition, the ruggedness of the landscape strongly suggests that the valleys defining POST-1, MS-I, MS-II, and POST-2 are themselves composed of a multiplicity of smaller valleys separated by barriers even smaller than that separating MS-I from MS-II. Thus, POST-1, MS-I, MS-II, and POST-2 are each expected to be composed of an ensemble of conformations, with the population of any one member of the ensemble depending on the exact depth of its valley and heights of the barriers separating it from its neighbors. As experimentally demonstrated in Figure 5, the depth of the valleys within POST-1, MS-I, MS-II, and POST-2, as well as the depths of the POST-1, MS-I, MS-II, and POST-2 valleys themselves, are sensitive functions of environmental conditions (e.g., substrate, cofactor, or allosteric effector binding) or the dissociation of reaction products. The circled numbers listed underneath the POST-1, MS-I, MS-II, and POST-2 valleys refer to the equivalently labeled POST, MS-I, and MS-II complexes depicted in Figure 3. Abbreviation: ΔG‡, free energy of activation.