Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly

Abstract

We report crystal structures of the Thermus thermophilus ribosome at 2.3- to 2.5-Å resolution, which have enabled modeling of rRNA modifications. The structures reveal contacts of modified nucleotides with mRNA and tRNAs or protein pY, and contacts within the ribosome interior stabilizing the functional fold of rRNA. Our work provides a resource to explore the roles of rRNA modifications and yields a more comprehensive atomic model of a bacterial ribosome.

Figure 1. Electron density maps allow comprehensive modeling of rRNA and ribosomal protein modifications of the 30S subunit

(a) Spatial distribution of modified nucleotides in the structure of the small ribosomal subunit from T. thermophilus. Here, in Fig. 2 and throughout the text rRNA nucleotides are named according to the E. coli numbering system. Panels (b-i) show unbiased difference F_o-F_c electron density maps for eight types of modifications present in 16S rRNA and ribosomal proteins S12 and S4. Grey mesh shows the F_o-F_c map after refinement with the entire modified nucleotides or amino acid residues omitted (contoured at 2.7-3.0σ). Green mesh shows the F_o-F_c electron density map after refinement with unmodified nucleotides (contoured at 1.5-2.5σ). The modifications shown are: (b) N3-methyluridine 1498, (c) N4,O2’-dimethylcytidine 1402, (d) N2-methylguanosine 1207, (e) N2-dimethylguanosine 966, (f) N7-methylguanosine 527, (g) N6-dimethyladenosine 1519; (h) β-methylthioaspartate of protein S12, (i) 4Fe-4S iron-sulfur cluster of protein S4.

Figure 2. Modeling of rRNA modifications of the 50S subunit

(a) Spatial distribution of modified nucleotides in the structure of the large ribosomal subunit from T. thermophilus. Panels (b-h) show unbiased difference F_o-F_c electron density maps for eight types of modifications present in 23S rRNA. The color scheme is the same as in Fig.1. The modifications shown are: (b) pseudouridine 2605, (c) 5-methyluridine 1939, (d) 2’-O-methyluridine 2552, (e) 5-methylcytidines 1942 and 1962, (f) 2’-O-methylguanosine 2251; (g) 2’-O-methylcytidine 1920, (h) 2-methyladenosine 2503.

Figure 3. Modified nucleotides form numerous molecular contacts with the ribosome ligands and within the ribosome interior

(a) Categorization of modified rRNA nucleotides into three groups based on their molecular contacts: (i) interacting with the ribosome ligands (orange), (ii) forming the inter-subunit bridges (light blue), and (iii) strengthening RNA-RNA and RNA-protein interactions within ribosomal interior (yellow). The actual sites of modifications are shown as red spheres. Modifications specific only to T. thermophilus are labeled in red (see Supplementary Table 2). (b-c)rRNA modifications that directly interact with either mRNA and tRNAs (b), or with protein pY (c). (d) rRNA modifications that maintain structure of the helix 44 in the mRNA channel (mRNA is not shown for clarity). In panels (b-d), modified residues of 16S rRNA are shown in orange, unmodified residues are in in light yellow.