Protein-guided RNA dynamics during early ribosome assembly

Abstract

The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the rRNA structure so that later proteins may join the complex is poorly understood. Here we use single molecule fluorescence resonance energy transfer (smFRET) to observe real-time encounters between ribosomal protein S4 and the 16S 5′ domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free energy path to protein-RNA recognition. Three-color FRET and molecular dynamics (MD) simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. This protein-guided dynamics offers an alternative explanation for induced fit in RNA-protein complexes.

Biophysics of protein evolution and evolutionary protein biophysics

“…Protein structures are dynamic; and conformational dynamics is crucial in many biomolecular interactions [171,172]. Even for globular proteins that fold to an essentially unique native structure under physiological conditions, other less favourable 'excited-state' conformations are always populated, albeit to a much lesser extent than the dominant native conformation that is commonly identified as the ground-state structure.…”

Differential effects of ribosomal proteins and Mg2+ ions on a conformational switch during 30S ribosome 5′-domain assembly

“…1A; Stern et al 1986;Gerstner et al 2001;Bellur and Woodson 2009) and nucleates in vitro assembly of the 5 ′ and central domains (Nowotny and Nierhaus 1988). Time-resolved footprinting and smFRET experiments showed that S4 binding induces a series of conformational changes in the 5WJ, leading to the native S4-rRNA complex (Mayerle et al 2011;Kim et al 2014). Remarkably, S4 binding also stabilizes tertiary interactions throughout the 5 ′ -domain RNA, which includes interactions as much as 60 Å away from the S4 binding site.…”

“…Previous smFRET experiments in which the 5 ′ -domain RNA was tethered to a microscope slide showed that higher Mg 2+ concentrations favor the docked (native) complex (Kim et al 2014). These experiments only counted long-lived complexes that could be captured on the slide.…”

“…We established a FRET assay to measure the populations of native and non-native (intermediate) S4-rRNA complexes (Kim et al 2014) at varying Mg 2+ concentrations and in the presence of different 5 ′ -domain proteins. In the mature 30S ribosome, 16S helix 3 docks under a pseudoknot in helix 18 (530 loop), resulting in efficient energy transfer from a Cy3 donor placed near the end of helix 3 and a Cy5 acceptor on protein S4 (Fig.…”

Structural and mechanistic insights into human splicing factor SF3b complex derived using an integrated approach guided by the cryo-EM density maps

“…Therefore, these proteins, especially SF3b49 and SF3b145, can possibly act as RNA chaperones 96,97 for the U12 snRNA to take up the right tertiary structure, analogous to that observed in the case of ribosomal proteins for rRNA. [98][99][100] Earlier, extensive mutational and deletion analysis of the SLI (14-17 nucleotides) and single stranded linker region (24-31 nucleotides) of U12 snRNA have shown that they affect splicing. So these regions have been implicated to be spacer elements or distance constraints in the functional form of U12 snRNA, such as during its interaction with pre-mRNA.…”