Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2

Abstract

Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function.

Figure 1.

Secondary structure of domain IV of E. coli 23S rRNA. (A) H66 is boxed in a dotted line. Bars indicate Watson–Crick-type base pairs, black dots indicate wobble base pairs. Non-canonical base pairs are shown in circles. Bases positions selected as compensatory mutations are circled, with the mutation indicated by an arrow. (B) Stem loop sequence of H66. The sequences randomized for SSER are boxed. Deletion mutations tested in this study are also indicated. (C) Tertiary structure of the internal bulge region (N₆) of H66. The structure of the base-triple, composed of C1800, G1817 and A1819, is shown in red. The other three bases that form the core structure are shown in pink. H-bonds are indicated by dotted lines. Coordinates were obtained from 2AW4 (5).

Figure 2.

Sequences of the genetically selected functional variants of the N₆-region in H66. (A) Secondary structures of the consensus class I L2-binding site, and the 11 class I variants. Consensus base-triple nucleotides (C1800, G1817 and A1819) in each variant are boxed. The inset shows the chemical structure of the base-triple. (B) Secondary structures of the consensus class II L2-binding site, and the 9 class II variants. Consensus base-triple nucleotides (A1800, U1817 and A1819) in each variant are boxed. The inset shows the chemical structure of the base-triple.

Figure 3.

Subunit association profiles of the H66 variants. (A) Sucrose density gradient centrifugation profiles of wild-type ribosomes, and the H66 variants, in the presence of 6 mM Mg²⁺. Peak assignments for the 70S, 50S and 30S subunits are indicated in the wild-type profile. (B) Sucrose density gradient centrifugation profiles of the N₆-16 revertants, with the indicated mutations (top two panels), and wild-type ribosomes carrying the indicated point mutation (bottom two panels).

Figure 4.

Molecular interactions between L2 and H66 in E. coli 50S subunit. (A) Structures of H66 (cyan), H64 (gray) and H34 (orange) interacting with L2 (green). The L2-binding site (N₆ region) is colored in red and pink, as shown in Figure 1C. The positions of G729, C1790 and G1989 are indicated. The intersubunit bridges B7b (L2), B4 (H34) and B5 (H64) are shown in blue. Coordinates were obtained from 2AW4 (5). (B) Enlarged view of the H34–H66 interaction through G729–C1774 pairing. C1790 and two residues (K206 and V16) in L2 are also involved in this interaction.