Structures of tRNAs with an expanded anticodon loop in the decoding center of the 30S ribosomal subunit

Abstract

During translation, some +1 frameshift mRNA sites are decoded by frameshift suppressor tRNAs that contain an extra base in their anticodon loops. Similarly engineered tRNAs have been used to insert nonnatural amino acids into proteins. Here, we report crystal structures of two anticodon stem-loops (ASLs) from tRNAs known to facilitate +1 frameshifting bound to the 30S ribosomal subunit with their cognate mRNAs. ASL(CCCG) and ASL(ACCC) (5'-3' nomenclature) form unpredicted anticodon-codon interactions where the anticodon base 34 at the wobble position contacts either the fourth codon base or the third and fourth codon bases. In addition, we report the structure of ASL(ACGA) bound to the 30S ribosomal subunit with its cognate mRNA. The tRNA containing this ASL was previously shown to be unable to facilitate +1 frameshifting in competition with normal tRNAs (Hohsaka et al. 2001), and interestingly, it displays a normal anticodon-codon interaction. These structures show that the expanded anticodon loop of +1 frameshift promoting tRNAs are flexible enough to adopt conformations that allow three bases of the anticodon to span four bases of the mRNA. Therefore it appears that normal triplet pairing is not an absolute constraint of the decoding center.

FIGURE 1.

Secondary structure of anticodon loops with corresponding cognate mRNAs that were complexed to the 30S A site. (A) Cognate anticodon–codon interaction between a normal ASL, ASL_GAA, and mRNA UUU. (B–D) ASLs and mRNAs used in this study. These ASLs all contain eight rather than seven bases in their anticodon loop. The additional base is shown in bold.

FIGURE 2.

Comparison of a normal anticodon–codon interaction and extended anticodon–codon interactions in the 30S A site. Left panels show the mRNA on the left with the ASL anticodon located on the right. Right panels depict 180° vertical reorientations of the complexes, displaying the interactions of 16S rRNA bases A1492, A1493, G530, and C1054 (in gray) with the codon–anticodon helix. For C, E, and G, the final 3fo–2fc map is cut at 5 Å radius and displayed at 1.8 sigma. (A,B) Normal anticodon–codon interaction between ASL_GAA (lime green) and its mRNA (pink) (Ogle et al. 2001). (C,D) ASL_CCCG (blue) interaction with its cognate mRNA (red). (E,F) ASL_ACCC (cyan) interaction with its cognate mRNA (magenta). (G,H) ASL_ACGA (green) interaction with its cognate mRNA (orange).

FIGURE 3.

Toeprint assay of translocation of tRNA_ACGA. Each band corresponds to a defined position of the ribosome on the mRNA, produced from a DNA oligonucleotide primer annealed to the mRNA upstream of the ribosome and extended by reverse transcriptase until it reaches the ribosome and falls off. The arrows indicate the position of the toeprint band corresponding to the mRNA codon in the P site. Positioning of the UCG codon in the P site would correspond to three-base translocation while UCGU in the P site corresponds to four-base translocation from the AUG codon. (Lane 1) mRNA positioning by direct P site binding of tRNA_ACGA. (Lane 2) mRNA positioning by P-site binding of tRNA_CAU and addition of tRNA_ACGA in the A site. (Lane 3) Same as lane 2 but after addition of EF-G + GTP.