Differences in Human and Bacterial Ribosome Decoding

Ribosomes are important molecular machines in the cell that synthesizes proteins by precisely decoding the nucleotide sequence of messenger RNA (mRNA) using amino aminoacyl-tRNA substrates. By performing mechanistic studies of bacterial and human ribosomes, researchers can understand their similarities and differences to develop drugs and understand diseases. On April 5, 2023, a team of scientists at St. Jude Children's Research Hospital published a research paper titled "mRNA decoding in human is kinetically and structurally distinct from bacteria" in the journal Nature. The study combines single-molecule imaging and cryo-electron microscopy approaches to examine the molecular basis of ribosomal fidelity in humans, revealing that the decoding mechanism differs dynamically and structurally from bacteria.

The translation of mRNA into protein is accomplished by the subunit ribosome, and the evolutionarily conserved core regions of ribosomal subunits in different species are associated with the interaction of structurally similar but sequence-diverse aminoacyl-tRNA adaptor molecules. Emerging human therapies utilize targeted mRNA decoding processes to treat disease. The decoding process involves initial selection and peptide bond formation, which determine the fidelity of decoding through proofreading selection. The current understanding of the decoding mechanism comes primarily from studies of bacterial systems. Although key features are retained during evolution, eukaryotes achieve higher fidelity of mRNA decoding than bacteria. In addition, structural snapshots of mammalian ribosomes isolated from cell extracts, as well as recent tomography studies, suggest that mammalian ribosomes undergo a subunit "rolling" process that is not observed in bacteria.

Researchers used multi-view single-molecule fluorescence resonance energy transfer (smFRET) imaging to study the kinetics and structural dynamics of human decoding. The results showed that the binding rate of the ternary complex to human ribosomes was about two-fold lower compared to bacteria. In addition, most homologous aa-tRNA binding events are productive, whereas the major pathway in bacteria is non-productive dissociation. Importantly, the intermediate-to-high-FRET transition prior to PRE complex formation is approximately 10-fold slower compared to bacteria. These findings suggest that the slowness of human decoding is due to physical conformational differences with bacteria.

Fig. 1 Domain closure and initial ternary complex binding.¹

The study found that although humans and bacteria both decode mRNA, the movement pathway of aminoacyl-tRNA during decoding is dissimilar and much slower in the human ribosome. These disparities stem from structural components present in the human ribosome and human elongation factor eEF1A, which collaborate to ensure the accurate integration of appropriate tRNA with every mRNA codon. The different nature and timing of conformational changes within the ribosome and eEF1A may be responsible for the higher decoding accuracy achieved by the human ribosome. Finally, the scientists also analyzed the binding site of the ribosome inhibitors, and these results are expected to help improve the clinical efficacy of each small molecule in the treatment of human diseases.

Fig. 2 Binding sites for ribosome inhibitors.¹

In summary, the study shows that conformational changes in the ribosome and eEF1A, as well as the physical properties of the incoming aa-tRNA molecule, together regulate decoding. The researchers also identified the steps that slow down the decoding process in the human ribosome. The ribosome selects the correct tRNA in a two-step process: initial selection and proofreading selection. Proofreading selection is where the ribosome checks for the second time that it has selected the correct molecule, and this is the step where human decoding is 10 times slower than that of bacteria. In addition, several drugs were found to target the proofreading selection process rather than the initial selection. Thus, these drugs did not hit similar steps between humans and bacteria. These findings have important implications for therapy development and cellular regulation.

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Reference

1. Holm, Mikael, et al. "mRNA decoding in human is kinetically and structurally distinct from bacteria." Nature 617.7959 (2023): 200-207.