Global Stabilization of rRNA Structure by Ribosomal Proteins S4, S17, and S20

Abstract

SummaryRibosomal proteins stabilize the folded structure of the rRNA and enable the recruitment of further proteins to the complex. Quantitative hydroxyl radical footprinting was used to measure the extent to which three different primary assembly proteins, S4, S17 and S20, stabilize the 3D structure of the E. coli 16S 5′ domain. The stability of the complexes was perturbed by varying the concentration of MgCl 2 . Each protein influences the stability of the rRNA tertiary interactions beyond its immediate binding site. S4 and S17 stabilize the entire 5′ domain, while S20 has a more local effect. Multi-stage folding of individual helices within the 5′ domain shows that each protein stabilizes a different ensemble of structural intermediates, that include non-native interactions at low Mg 2+ . We propose that the combined interactions of S4, S17 and S20 with different helical junctions bias the free energy landscape toward a few RNA conformations that are competent to add the secondary assembly protein S16 in the next step of assembly. Keywordsribosome; ribosomal protein; RNA folding; hydroxyl radical footprinting; RNA-protein interactions Ribosome biogenesis is one of the most important synthetic tasks undertaken by the cell. Among bacteria, short generation times require that several hundreds of ribosomes are produced per minute in order to produce the tens of thousands of ribosomes needed by each new cell 1;2 . A critical question is how the RNA and protein components of the ribosome interact to achieve the native subunits and avoid nonfunctional complexes.Studies on the reconstitution of the 30S ribosomal subunit have shown that binding of ribosomal proteins is coupled to folding of the rRNA 3;4 . The addition of the primary assembly proteins, which bind directly to the 16S rRNA, stabilize the helices with which they interact, and enable further assembly of each 30S domain by pre-ordering the binding sites for secondary and tertiary assembly proteins 5;6 . The extent to which individual ribosomal proteins stabilize the structure of the rRNA beyond their immediate binding site is thus directly connected to the hierarchy and cooperativity of assembly 4;7 .

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

“…In both models, Mg 2+ ions stabilize the native rRNA even in the absence of S4, as seen in footprinting experiments (Powers and Noller 1995b;Bellur and Woodson 2009;Ramaswamy and Woodson 2009a). The high-resolution crystal structure of the E. coli ribosome (Berk et al 2006; PDB ID 2I2P) reveals ordered, partially dehydrated magnesium ions coordinated with the right-angle junction between 16S helices 3 and 4 and the pseudoknot in helix 18.…”

“…To evaluate K 1 and K N , we used information from earlier hydroxyl radical footprinting experiments on the 16S 5 ′ -domain RNA that measured the fraction of free RNA in the native conformation (helix 3 docked) at different Mg 2+ concentrations and in the presence of different primary assembly proteins (Ramaswamy and Woodson 2009a). Estimates of K 1 from footprinting data allowed us to calculate binding constants K F , K N , and the linkage α = K N /K F = K 2 /K 1 , which describes how strongly S4 binding is coupled to docking of helix 3 compared to the free RNA.…”

“…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. Protein S17 interacts with 16S helix 11 and also indirectly stabilizes tertiary interactions in the lower half of the 5 ′ domain (Ramaswamy and Woodson 2009a). Binding of protein S20 to helices 6, 8, 9, and 10 stabilizes those helix junctions and mediates interactions with helix 44 later in 30S assembly (Dutca and Culver 2008;Ramaswamy and Woodson 2009a).…”

Ribosome Biogenesis in the YeastSaccharomyces cerevisiae

“…Binding of r-proteins is thought to stabilize productively folded rRNA conformers or function as chaperones to redirect misfolded rRNA (Semrad et al 2004;Kovacs et al 2009;Woodson 2012). Binding of r-proteins can alter RNA structure locally as well as distally to create binding sites for subsequently assembling r-proteins (Jagannathan and Culver 2004;Ramaswamy and Woodson 2009). In this way assembly in vitro may be driven by alternating and parallel series of rRNA folding and protein-binding steps.…”

Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly

“…The hierarchical addition of ribosomal proteins to the 16S rRNA (Held et al 1974) arises from conformational changes to the rRNA induced by early binding proteins. In some examples, primary assembly proteins capture the folded structure of the rRNA (Weeks and Cech 1996;Menichelli et al 2007), in turn stabilizing rRNA tertiary interactions and the binding sites of secondary assembly proteins (Agalarov and Williamson 2000;Ramaswamy and Woodson 2009a). Many ribosomal proteins also change structure when they join the complex, however, and the resulting mutually induced fit is expected to increase the specificity of assembly (Williamson 2000;Bokinsky et al 2006).…”