An Atlas of the base inter-RNA stacks involved in bacterial translation

Nucleobase-specific noncovalent interactions (i.e., inter-RNA base pairing and stacking) play a crucial role in the RNA-driven biochemical process of translation. Although the structural details of translation are well studied, especially base-pairing, the role of nucleobase-specific inter-RNA stacking interactions is still not fully appreciated. Herein, we provide a comprehensive analysis of the stacking interactions between different RNA components in the available crystal structures of the bacterial ribosome caught at different stages of translation. Analysis of tRNA||rRNA stacking interactions reveals distinct stacking behaviour; both the A-and E-site tRNAs exhibit unique stacking patterns with 23S rRNA bases, while P-site tRNAs stack with 16S rRNA bases. Furthermore, E-site stacks exhibit diverse face orientations and ring topologies―rare for inter-chain RNA interactions―with higher average interaction energies than found in either A or P-site rRNA||tRNA stacks. This suggests that stacking may be essential for stabilizing tRNA progression to and through the E-site. Additionally, examination of mRNA||rRNA stacking interactions reveals other stacking geometries, which depend on the site of tRNA binding; A-site mRNA||rRNA stacks exhibit more frequent interactions with high stability, suggesting they play an essential role in mRNA positioning within the translational complex. Similarly, analysis of 16S rRNA||23S rRNA stacks highlights the importance of specific bases in maintaining the integrity of the translational complex by linking the two rRNAs. Furthermore, tRNA||mRNA stacking interactions exhibit distinct geometries and energetics at the E-site, indicating their significance during tRNA translocation and elimination. Overall, the analysis demonstrates that both A and E-sites display a broader and more diverse distribution of inter-RNA stacking interactions compared to the P-site. Notably, P-site stacking interactions are the least stable, suggesting their diminished role due to the need for interactions to be dominated by codon:anticodon base pairing to avoid misreading the mRNA. Stacking interactions in the active ribosome are not simply accidental byproducts of biochemistry but are invoked to compensate and support the integrity and dynamics of translation.