Mapping of the second tetracycline binding site on the ribosomal small subunit of E.coli

Abstract

Tetracycline blocks stable binding of aminoacyl-tRNA to the bacterial ribosomal A-site. Various tetracycline binding sites have been identified in crystals of the 30S ribosomal small subunit of Thermus thermophilus. Here we describe a direct photo- affinity modification of the ribosomal small subunits of Escherichia coli with 7-[3H]-tetracycline. To select for specific interactions, an excess of the 30S subunits over tetracycline has been used. Primer extension analysis of the 16S rRNA revealed two sites of the modifications: C936 and C948. Considering available data on tetracycline interactions with the prokaryotic 30S subunits, including the presented data (E.coli), X-ray data (T.thermophilus) and genetic data (Helicobacter pylori, E.coli), a second high affinity tetracycline binding site is proposed within the 3'-major domain of the 16S rRNA, in addition to the A-site related tetracycline binding site.

Figure 1

(A) Structure of Tc complex with Mg²⁺ (25). (B) Absorption spectrum of Tc.

Figure 2

Primer extension analysis of the 16S RNA using the primer CGACAGCCATGCAGCACC complementary to G1047–G1064 of the 16S rRNA. Separation on an 8% polyacrylamide-urea gel demonstrates reverse transcriptase primer extension stops at positions A937 and A949, caused by modification of the 16S rRNA with Tc. The fragment of the 16S rRNA sequence A918–U957 is shown. Line 1, the 16S rRNA isolated from the irradiated Tc-30S subunit complex; line 2, the 16S rRNA isolated from irradiated 30S subunits (no Tc); line 3, the 16S rRNA isolated from 30S subunits (no irradiation).

Figure 3

Putative sites of Tc interactions with the 16S rRNA within crystals of the 30S ribosomal subunit of T.thermophilus according to PDB 1I97 (10). PDB data were analyzed with Swiss PDB Viewer 3.6b3 (http://cn.expasy.org/spdbv). The 16S rRNA sequence numbering is according to E.coli. G942 corresponds to in vivo genetic data (12), C936 and C948 data from this publication. The ribosomal small subunit interface with six putative Tc binding sites is on the left. (A) The extracted structure of the main sub-domain of the 3′-end major domain of the 16S rRNA (–28) showing RNA in the dark gray ribbons, and S7 protein in light gray cylinders. Tet-4, Tet-6 and G942, C936, C948 are shown. Orientation of the sub-domain is the same as for the subunit. (B) Space-filled Tet-4 and Tet-6 (black), and the 16S rRNA nucleotides (gray) are depicted with the same orientation as in (A). (C) The image is depicted at an orientation, different from that in (B), to show the distances (Å) in more detail.