Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function

Abstract

All organisms incorporate post-transcriptional modifications into ribosomal RNA, influencing ribosome assembly and function in ways that are poorly understood. The most highly conserved modification is the dimethylation of two adenosines near the 3' end of the small subunit rRNA. Lack of these methylations due to deficiency in the KsgA methyltransferase stimulates translational errors during both the initiation and elongation phases of protein synthesis and confers resistance to the antibiotic kasugamycin. Here, we present the X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit lacking these dimethylations. Our data indicate that the KsgA-directed methylations facilitate structural rearrangements in order to establish a functionally optimum subunit conformation during the final stages of ribosome assembly.

FIGURE 1.

Position of the modified nucleotides A1518 and A1519 in the 30S ribosomal subunit. (A) Secondary structure of T. thermophilus 16S rRNA (Cannone et al. 2002), indicating positions and sequences of helices 6, 24a, 44, and 45 that show structural changes in the unmethylated 30S subunit. (B) Positions of helices 6, 24a, 44, and 45 in the crystal structure of the T. thermophilus 30S subunit (Wimberly et al. 2000). Ribosomal proteins in panel B are omitted for clarity.

FIGURE 2.

Structural changes in the unmethylated 30S subunit. (A) Final σ_A-weighted 2mF_o − DF_c electron density in the ksgA mutant 30S subunit crystal structure. (Red) 16S rRNA helix 24a (h24a); (green) helix 44 (h44); (blue) helix 45 (h45); (violet) the GGAA tetraloop of helix 45. (B) Contacts between helix 45 and helices 24a and 44. Helices are colored as in A. (C) Bending of the GGAA tetraloop in the unmethylated subunit (violet) is visible. The wild-type 30S subunit structure (PDB entry 1J5E) (Wimberly et al. 2000) is superimposed (gray). Kasugamycin (Ksg, orange spheres) from PDB entry 1VS5 (Schuwirth et al. 2006) is superimposed. (D) Comparison of the helix 45 tetraloop with a GCAA tetraloop as observed in PDB entry 1ZIH (Jucker et al. 1996). (E,F) The ksgA mutation prevents the formation of a hydrogen-bonding network between helices 45 and 44. The helix 45–44 interface in the ksgA mutant (e.g., 30S subunits prior to methylation) (E) and in the wild-type, fully methylated 30S subunit (F). Panels E and F are from identical viewpoints.

FIGURE 3.

Structural readjustments of helices adjacent to the unmethylated GGAA tetraloop. Movement of A793 in the kasugamycin-binding site in unmethylated (A) and fully methylated (Schuwirth et al. 2006) (B) 30S subunits. (C) Close-up view illustrating shifts of A1492, A1493, and C1054 in the decoding site of the unmethylated 30S (cyan) in comparison with the wild-type (gray) subunit (PDB 1J5E) (Wimberly et al. 2000), colored as in Figure 2B. (D) Close-up view illustrating the shift in position of 16S rRNA helix 6 of the unmethylated 30S subunit (orange) in the P site of an adjacent 30S subunit.