How to remain flexible: the versatile active site of the ribosome
The ribosome is an ancient ribozyme that can catalyze a variety of chemical reactions. Researchers at the Max Planck Institute for Biophysical Chemistry in Göttingen have now explored the mechanisms of the ribosome’s two main activities by determining the number of protons "in flight" in the transition state. They demonstrate that one active site, with a small difference introduced by a few amino acids of a protein called release factor, can support rather different reaction mechanisms. This unique versatility is probably due to the fact that ribosomal residues, and residues of the release factor, do not take part in chemistry, but rather provide a network of electrostatic and hydrogen-bonding interactions that help to orient and stabilize the substrates and transition states, more or less independent of their chemical nature. This flexibility of the ribosome’s active site may explain its ability to catalyze a number of various chemical reactions and presumably played an important role through evolution.
Life began when RNA emerged from a prebiotic soup of chemicals. At the early stages of evolution, in the primordial "RNA World", RNA combined the functions of storage and transmission of genetic information and catalysis, enabling both replication and metabolism. The RNA world gave rise to today's world of DNA, RNA, and protein, in which the catalytic functions have been taken over by protein enzymes, and the chemically more stable DNA replaced RNA as the preferred genetic material. Owing to the generally higher catalytic efficiency of protein enzymes, only a few RNA enzymes have survived through evolution. Modern ribozymes are highly specialized molecules that usually catalyze phosphoryl transfer reactions, activating a 2'-OH group of a ribose residue within the RNA or a water molecule for the nucleophilic attack on a phosphodiester bond. The only notable exception is the ribosome, a large ribonucleoprotein particle that synthesizes proteins in all cells. The active site of the ribosome is built of RNA, making it the largest modern ribozyme, the only one that has at least two activities: a polymerase activity to make polypeptides from aminoacyl-tRNAs and a hydrolytic activity to release the completed peptide from its tRNA carrier. Furthermore, experiments using artificial substrates revealed an even higher versatility, showing that the ribosome in addition to making peptide bonds could also support the formation of ester, thioester, thioamide, or phosphinoamide bonds. Thus, the question arises as to how the same active site can catalyze all these different reactions and whether the catalytic mechanisms, as represented by the transition states of the reactions, are the same or different. A recent study by Göttingen Max Planck researchers Stephan Kuhlenkötter, Wolfgang Wintermeyer, and Marina V. Rodnina published in Nature offers a possible solution.
The active site of the ribosome, the peptidyl transferase center, is located on the large ribosomal subunit within a highly conserved region of the ribosomal RNA (23S rRNA in bacteria) (Fig. 1A). The primordial rRNA is believed to have been a rather small molecule, which originally was formed by a duplication of a core element to form a pseudo-symmetrical active site (Fig. 1B) and gradually expanded to the modern size through the addition of new elements. In the active site, the CCA ends of the tRNA substrates are bound in an orientation that allows the alpha-amino group of aminoacyl-tRNA to attack the ester bond of peptidyl-tRNA to form a peptide bond. The same active site, augmented by residues of a protein factor called release factor, supports the hydrolysis of peptidyl-tRNA, this time activating a water molecule for nucleophilic attack. The two reactions require that one or more protons move during the reactions. Different transition states may be distinguished by the number of protons "in flight", i.e. protons transferred in the transition state. Determining this number can provide insight into the molecular mechanisms.