How R-Proteins Stabilize the R-RNA during Early Stages of Bacterial Ribosome Biogenesis

Abstract

Ribosome biogenesis in E. coli has been extensively studied, yet, very little is known about the structural changes that occur to the rRNA upon r-protein binding. Recent cryo-electron microscopy and mass spectrometry studies from the Williamson laboratory have produced a time-resolved assembly map of the 16S subunit. The first r-protein to nucleate 16S folding, S4, binds to h16-h18 of the five-way junction–a signature region that idiosyncratically separates Bacteria and Archaea. S4, subsequently, nucleates the formation of the 5' domain of the 16S and recruits other r-proteins to bind.Guided by in vitro studies, we probe how S4 nucleates the 5' domain formation using a series of all-atom molecular dynamics (MD) simulations on the E. coli 5' domain with various r-proteins systematically bound to the rRNA. Large dynamic fluctuations in the structure of the rRNA were observed in both our long-time MD trajectories and multi-color single molecule FRET experiments from our collaborators. For example, with only S4 bound to the rRNA, an A-minor interaction between h8 and h14 opens up, but is stable upon adding S17 to a location distant from h8 and h14.We also are exploring the cooperative effects of the r-protein binding by generating weighted networks based on position correlations. Graph of the network weighed by edge-betweenness, a measure of the number of shortest paths that pass through a given edge, show a continuous path connecting the binding sites of S4, S17, and S20 with a branching point located near h15–the binding site the secondary binding r-protein S16. This path is hypothesized to modulate the binding between primary and secondary binding r-proteins and further probed using additional all-atom and structure-based models.