Visualization of protein S1 within the 30S ribosomal subunit and its interaction with messenger RNA

Abstract

S1 is the largest ribosomal protein, present in the small subunit of the bacterial ribosome. It has a pivotal role in stabilizing the mRNA on the ribosome. Thus far, S1 has eluded structural determination. We have identified the S1 protein mass in the cryo-electron microscopic map of the Escherichia coli ribosome by comparing the map with a recent x-ray crystallographic structure of the 30S subunit, which lacks S1. According to our finding, S1 is located at the junction of head, platform, and main body of the 30S subunit, thus explaining all existing biochemical and crosslinking data. Protein S1 as identified in our map has a complex, elongated shape with two holes in its central portion. The N-terminal domain, forming one of the extensions, penetrates into the head of the 30S subunit. Evidence for direct interaction of S1 with 11 nucleotides of the mRNA, immediately upstream of the Shine-Dalgarno sequence, explains the protein's role in the recognition of the 5' region of mRNA.

Figure 1

Location of S1 within the 30S subunit. (a) Surface representation of the 11.5-Å resolution cryo-EM map (29). (b) X-ray structure of the 30S subunit (25), filtered to the resolution of the cryo-EM map and shown in the same solvent-side orientation. The area highlighted with a rectangle shows a large, extra mass of density in the cryo-EM map (a). (c and d) The difference map (red), obtained by subtracting the masses corresponding to only 30S ribosomal proteins in the x-ray (25) and cryo-EM (31) maps, is superimposed on the cryo-EM map (c) and on the filtered x-ray map of the 30S subunit (d). In c, the 30S map is shown as a semitransparent surface, in which the difference density map is embedded. The S21 mass is marked with an arrow in c and d.

Figure 2

Neighborhood and structure of protein S1. (a) Position of protein S1 with respect to the16S rRNA, and (b) with respect to other small-subunit ribosomal proteins of the 30S x-ray structure (25). Components of the x-ray structure were filtered to the resolution of the S1 map. Some of the relevant 16S-rRNA helices and small-subunit proteins are labeled. c and d are stereo representations of S1 mass in two orientations: c showing the smaller hole; and d showing the bigger hole within the central globular (CG) region. SA and LA identify the short and long arms, respectively, of S1.

Figure 3

Molecular environment of S1. S1 is shown in red. (a and b) Neighboring proteins. (c and d) Neighboring16S-rRNA helices. Atomic coordinates were taken from Wimberly and coworkers (25) and are represented by using RIBBONS (54). Boxed areas to the right of each panel represent the 30S x-ray structure in the corresponding views, as an aid to orientation. The 30S subunit is viewed from the solvent side (a and c) and from the 50S-interface side (b and d). The bigger hole within the CG portion of the S1 mass is clearly visible in both views. Landmarks are the same as in Fig. 2.

Figure 4

Stereo presentation of the relative positions of S1, helix 45 of 16S rRNA, and mRNA segment. The mRNA segment (orange) was derived from a previous study (32) by computing a difference map between cryo-EM maps of ribosomes with and without bound mRNA and by fitting the atomic coordinates of a single-stranded 12-nucleotide stretch by using polypyrimidine. The mRNA segment corresponding to the 11-nucleotide stretch is seen embedded on the surface of the CG region of the S1 (see text). The 3′ end of the mRNA lies near the 3′ end of helix 45 (blue), from which the anti-SD sequence extends. The actual anti-SD region of the 16S rRNA is not shown because of conflicting positions in the x-ray maps of the isolated 30S subunit (25) and the 70S ribosome (53) (see text). The S1 mass is shown as semitransparent red. Landmarks are the same as in Fig. 2.