Conserved Loop Sequence of Helix 69 in Escherichia coli 23 S rRNA Is Involved in A-site tRNA Binding and Translational Fidelity

Abstract

Ribosomes translate the genetic information contained in mRNAs into proteins. The large (50 S) subunit of the ribosome catalyzes the formation of a peptide bond between the aminoacyl-tRNA (aa-tRNA) 3 bound to the A-site and the peptidyl-tRNA at the P-site. This peptide bond formation takes place at the peptidyltransferase center of the 50 S subunit. Codon-anticodon pairing occurs at the decoding center of the small (30 S) subunit. The aa-tRNA is delivered to the ribosome as a ternary complex of aa-tRNA, EF-Tu, and GTP. Cognate codon recognition is strictly monitored by 16 S rRNA and triggers GTP hydrolysis and dissociation of EF-Tu. This allows aa-tRNA to be accommodated by the A site of the 50 S subunit. Thus, accuracy of protein synthesis is based on the synergistic interplay of the large and small subunits of the ribosome. However, mechanistic insights into the ribosome dynamics during decoding are still rudimentary.The intersubunit bridges of the ribosome are functional sites that are not only necessary for subunit connection but also play roles in translation. Helix 69 (H69) (position 1906 -1924) is a highly conserved stemloop in domain IV of 23 S rRNA of the bacterial 50 S subunit (Fig. 1A). In fact, each base in the loop of H69 shows more than 98% conservation in 436 bacterial rRNAs (www.rna.icmb.utexas.edu). Crystallographic studies revealed that H69 is located on the surface involved in intersubunit association with the 30 S subunit by connecting with helix 44 (h44) of 16 S rRNA forming the bridge B2a; A1912, A1913, A1914, A1918 and A1919 in H69 make contact with positions 1407-1410 and 1494 -1495 in h44 and G1517 in h45 (1, 2) (see Fig. 7A). In the free 50 S subunit, H69 makes a compact structure and interacts with H71 of 23 S rRNA (3), whereas in the 70 S ribosome H69 stretches toward the small subunit and interacts with h44 of 16 S rRNA (1). The tip of H69 moves about 13.5 Å during this structural change. In addition, H69 directly interacts with A/T-, A-, and P-site tRNAs during each translation step (1). During the decoding step, aa-tRNA is brought into the A/T-site as a complex with EF-Tu/GTP (ternary complex). Cryoelectron microscopy analyses showed a kinked conformation for aa-tRNA at the A/T state (molecular spring) (4, 5). The tip of H69 makes a contact with the hinge of the kink between D-and anticodon-stems in tRNA. This interaction is supposed to facilitate the structural distortion of tRNA that enables the anticodon-stem to fit into the decoding center of 16 S rRNA. In the crystal structure of the 70 S ribosome complexed to both A-and P-site tRNAs, H69 is positioned between the two tRNAs (1). The minor groove of H69 (positions 1908 -1909, 1922-1923) interacts with the minor groove of the D-stem of the P-site tRNA (positions 12-13, 25-26) (Fig. 1B), whereas the conserved loop (positions 1913-1915) of H69 makes a contact with the D-stem of A-site tRNA (positions 11-12 and 25-26) (Fig. 1B). Moreover, it has been reported that H69 interacts with various translational factors. In the post-t...

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