Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits

Abstract

The 18S rRNA of the small eukaryotic ribosomal subunit contains several expansion segments. Electron microscopy data indicate that two of the largest expansion segments are juxtaposed in intact 40S subunits, and data from phylogenetic sequence comparisons indicate that these two expansion segments contain complementary sequences that could form a direct tertiary interaction on the ribosome. We have investigated the secondary structure of the two expansion segments in the region around the putative tertiary interaction. Ribosomes from yeast, wheat, and mouse-three organisms representing separate eukaryotic kingdoms-were isolated, and the structure of ES3 and part of the ES6 region were analyzed using the single-strand-specific chemical reagents CMCT and DMS and the double-strand-specific ribonuclease V1. The modification patterns were analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The investigated sequences were relatively exposed to chemical and enzymatic modification. This is in line with their indicated location on the surface at the solvent side of the subunit. The complementary ES3 and ES6 sequences were clearly inaccessible to single-strand modification, but available for cleavage by double-strand-specific RNase V1. The results are compatible with a direct helical interaction between bases in ES3 and ES6. Almost identical results were obtained with ribosomes from the three organisms investigated.

FIGURE 1.

Schematic illustration of the positions of expansion segments ES3 (black) and ES6 (dark gray) on the yeast 40S ribosomal subunits (Spahn et al. 2001) and in the secondary structure model of 18S rRNA (Comparative RNA Web site; http://www.rna.icmb.utexas.edu/; Cannone et al. 2002). The 40S subunit is viewed from the interface side (left) and from the solvent side (right). (Insets) Secondary structure models for yeast ES3 and ES6 (The European ribosomal RNA database, http://oberon.rug.ac.be:8080/rRNA/; Wuyts et al. 2002). The nucleotides are numbered in accordance with Figure 2 ▶. The proposed tertiary interaction between sequences in yeast ES3 and ES6 is based on the consensus sequences (Alkemar and Nygard 2003).

FIGURE 2.

Secondary structure data obtained for ES3 and ES6 in isolated yeast (A), wheat (B), and mouse (C) ribosomes. Native ribosomes isolated from yeast, wheat, and mouse were incubated in the presence of single-strand-specific reagents CMCT, final concentration 50 mM (blue), and DMS, final concentration 20 mM (red), or in the presence of the double-strand-specific ribonuclease V1, final concentration 1 unit/50 μL (green), as described in Materials and Methods. The generated modification patterns were analyzed using primer extension and gel electrophoresis in an ABI 377 automated DNA sequencer as described in Materials and Methods. Control samples (black) were incubated in the absence of modifying reagents but otherwise treated identically to the modified samples. The reagent-independent termination products seen in the control samples were used as internal standards for alignment and for adjusting the peak height for each of the individual overlaid lanes. Sequences are given in the 5′-to-3′ direction.

FIGURE 3.

Secondary structure models for ES3 and the 3′-part of ES6 in yeast (A), wheat (B), and mouse (C). The secondary structure models were generated using the Vienna RNA package (Hofacker et al. 1994). Sequence data were taken from GenBank accession nos. J01353, AY049040, and X00686 for Saccharomyces cerevisiae, Triticum aestivum, and Mus musculus, respectively. (•) Bases available for modification by single strand reagents; (arrows) phosphate bonds accessible to cleavage by the double-strand-specific RNase V1; (ns) reagent-independent stops. (D) Structural model of ES3 (blue), the ES6 structural elements helix H23_14, and its apical loop (green), with the bases proposed to participate in the tertiary interaction (red). The 3D model of ES3 and ES6 was constructed using ERNA–3D (Mueller et al. 1995).