Single Molecule Views of the Ribosome Assembly

Abstract

Rapid and accurate assembly of the ribosome is a fundamental requisite for cellular functions. Ribosome assembly is a complex but highly selective and coordinated process of RNA folding induced/accompanied by binding of ribosomal proteins. Cryo-EM and footprinting studies have provided the hierarchical map of the assembly and molecular details of the folding pathways. Yet, the dynamics during ribosome assembly, despite their potential importance in selectivity and coordination, are not well understood. Using single molecule fluorescence resonance energy transfer, we could reveal an early phase of ribosome assembly dynamics by focusing on the 5’ RNA domain of the small subunit. The RNA alone is highly dynamic and exhibits heterogeneous fluctuations. In particular, helix 3 undergoes extensive movement consistent with its switching mechanism proposed earlier based on footprinting data. Binding of the protein S4 reduces fluctuations but not completely, and binding of additional proteins further suppresses fluctuations and reduces heterogeneity as well. Interestingly, S4 protein binds to one conformation preferentially before switching to the other conformation that dominates in equilibrium, supporting the induced fit mechanism of S4 binding to RNA. Extending our approach to multiple colors should reveal the coordinated interplay of RNA folding and conformational dynamics with successive binding of proteins during ribosome assembly.