Genetic and structural analysis of base substitutions in the central pseudoknot of Thermus thermophilus 16S ribosomal RNA

Abstract

Characterization of base substitutions in rRNAs has provided important insights into the mechanism of protein synthesis. Knowledge of the structural effects of such alterations is limited, and could be greatly expanded with the development of a genetic system based on an organism amenable to both genetics and structural biology. Here, we describe the genetic analysis of base substitutions in 16S ribosomal RNA of the extreme thermophile Thermus thermophilus, and an analysis of the conformational effects of these substitutions by structure probing with base-specific modifying agents. Gene replacement methods were used to construct a derivative of strain HB8 carrying a single 16S rRNA gene, allowing the isolation of spontaneous streptomycin-resistant mutants and subsequent genetic mapping of mutations by recombination. The residues altered to give streptomycin resistance reside within the central pseudoknot structure of 16S rRNA comprised of helices 1 and 27, and participate in the U13-U20-A915 base triple, the G21-A914 type II sheared G-A base pair, or the G885-C912 Watson-Crick base pair closing helix 27. Substitutions at any of the three residues engaged in the base triple were found to confer resistance. Results from structure probing of the pseudoknot are consistent with perturbation of RNA conformation by these substitutions, potentially explaining their streptomycin-resistance phenotypes.

FIGURE 1.

(A) Location of the central pseudoknot (blue spheres) and the streptomycin (red) binding site in the T. thermophilus 30S subunit crystal structure (Carter et al. 2000). (B) Structure of the central pseudoknot, showing the U13–A915–U20 base triple (blue), the G21–A915 base pair (green), and the C912–G885 base pair (purple). Streptomycin is shown as red sticks with van der Waals radii indicated. (C) Secondary structure model of the central pseudoknot region of T. thermophilus HB8 16S rRNA (Cannone et al. 2002) modified to reflect the base pairing and base triple arrangements observed in the 30S subunit crystal structure (Wimberly et al. 2000). Sites of base substitutions conferring streptomycin resistance are indicated. (D) The U13–U20–A915 base triple and streptomycin (Sm), showing the hydrogen bonding network (dashed lines) and the site of DMS hyperreactivity (arrowhead) at the N1 of A915. (E) The G21–A914 type II sheared G–A base pair and streptomycin. The arrowhead indicates the N7 of A914 that becomes reactive to DMS in the A914G mutant. (F) The C912–G885 Watson–Crick pair and streptomycin. (A,B,D–F) Generated with PyMol (DeLano 2002) using PDB file 1FJG (Carter et al. 2000).

FIGURE 2.

Construction of the ΔrrsA∷ htk1 knockout strain. (A) The 16S rRNA gene rrsA is represented by the black box, with the surrounding chromosomal regions in white. The htk gene is represented by the gray box. Arrows indicate the direction of transcription. Dashed lines indicate the limits of sequence identity between the two chromosomal loci. Arrowheads indicate the positions of primers used for diagnostic PCR analysis; A, primer Tth P16S-1; B, primer Tth T16S-3; C, primer HTK-1; D, primer HTK-2. (B) Diagnostic PCR analysis of the chromosomal rrsA locus of HB8 and HG 286. L, size markers; wt, DNA from wild-type HB8; Δ, DNA from the ΔrrsA∷ htk1 mutant HG 286. Predicted sizes of the PCR products in base pairs are indicated below the gel.

FIGURE 3.

Structure probing of wild-type and mutant ribosomes in the helix 27 region of 16S rRNA with DMS. Wild-type ribosomes (lane 1); U13C (lane 2); U20G (lane 3); C912A (lane 4); A914G (lane 5); and A915G (lane 6). K, unmodified ribosomes; DMS, modification with dimethylsulfate; DSM–N7, modification with dimethylsulfate followed by reduction with NaBH₄ and cleavage with aniline to detect modification at the N7 of Gs. Arrowheads indicate the positions of C912, A914, and A915.