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

Abstract

Ribosomal protein S4 nucleates assembly of the 30S ribosome 5 ′ and central domains, which is crucial for the survival of cells. Protein S4 changes the structure of its 16S rRNA binding site, passing through a non-native intermediate complex before forming native S4-rRNA contacts. Ensemble FRET was used to measure the thermodynamic stability of non-native and native S4 complexes in the presence of Mg 2+ ions and other 5 ′ -domain proteins. Equilibrium titrations of Cy3-labeled 5 ′ -domain RNA with Cy5-labeled protein S4 showed that Mg 2+ ions preferentially stabilize the native S4-rRNA complex. In contrast, ribosomal proteins S20 and S16 act by destabilizing the non-native S4-rRNA complex. The full cooperative switch to the native complex requires S4, S16, and S20 and is achieved to a lesser degree by S4 and S16. The resulting thermodynamic model for assembly of the 30S body illustrates how ribosomal proteins selectively bias the equilibrium between alternative rRNA conformations, increasing the cooperativity of rRNA folding beyond what can be achieved by Mg 2+ ions alone.

Evolution of protein-coupled RNA dynamics during hierarchical assembly of ribosomal complexes

“…Protein S16 itself markedly stabilized the docked (native) conformation (Fig. 1g, j, k ), consistent with our previous ensemble FRET results 21 . These conformational preferences illustrate how each ribosomal protein perturbs the energy landscape for rRNA folding.…”

“…2 ), addition of protein S17 increased the population of flipped intermediate (Fig. 1f, i ), in agreement with ensemble FRET studies 21 and footprinting of 5′ domain complexes 16 . S17 binding also shifted the average FRET efficiency of the high FRET state from E ~ 0.61 to 0.53 and the low FRET population from E ~ 0.33 to 0.11 (Fig.…”

Zinc-finger protein CNBP alters the 3-D structure of lncRNA Braveheart in solution

“…Saltdependent association can be critical for biological function 34 . In RNA polymers, divalent magnesium (Mg 2+ ) is essential for folding, higher-order interactions and function 35 . As such, sensitivity to Mg 2+ is an indicator of the presence of tertiary interactions.…”

“…This behavior is not unlike the SAM-I riboswitch, which also has welldefined secondary and tertiary structure (in particular, a highresolution crystal structure in the ligand-bound state), but is highly flexible in its apo state. In fact, many structured RNAs can adapt multiple conformations and are highly flexible 1,2,35 . For example, even with 300 kV cryo-electron microscopy (cryo-EM) using an energy filter, Zhang et al could obtain only a~9 Å resolution map of 30 kDa RNA (47 nucleotide dimer) 51 .…”

Size-variable zone in V3 region of 16S rRNA

“…[12][13][14][15] It is important to maintain the structure because there are segments that play essential roles in ribosome function. For instance, ribosomal proteins attach to 16S rRNA in a hierarchical order, which produces a cooperativity effect; [16][17][18][19] however, it is possible that structural variations would affect this process. In this respect, information concerning the size distribution of the different variable regions would contribute to identifying and locating mutable fractions within the molecule and evaluating if these affect the structure of the molecule.…”