How mutations in tRNA distant from the anticodon affect the fidelity of decoding

Abstract

The ribosome converts genetic information into protein by selecting aminoacyl tRNAs whose anticodons base-pair to an mRNA codon. Mutations in the tRNA body can perturb this process and affect fidelity. The Hirsh suppressor is a well-studied tRNA(Trp) harboring a G24A mutation that allows readthrough of UGA stop codons. Here we present crystal structures of the 70S ribosome complexed with EF-Tu and aminoacyl tRNA (native tRNA(Trp), G24A tRNA(Trp) or the miscoding A9C tRNA(Trp)) bound to cognate UGG or near-cognate UGA codons, determined at 3.2-Å resolution. The A9C and G24A mutations lead to miscoding by facilitating the distortion of tRNA required for decoding. A9C accomplishes this by increasing tRNA flexibility, whereas G24A allows the formation of an additional hydrogen bond that stabilizes the distortion. Our results also suggest that each native tRNA will adopt a unique conformation when delivered to the ribosome that allows accurate decoding.

Figure 1

Overview of miscoding mutations and Trp-tRNA^Trp bound in the A/T state. A) The traditional cloverleaf diagram of Trp-tRNA^Trp showing the locations of the miscoding mutations A9C (orange) and G24A (red). B) When bound to the ribosome along with EF-Tu, the aminoacyl-tRNA (green) adopts the distorted A/T conformation. Structures were also determined for Trp-tRNA^Trp containing mutations at position 24 (red) and 9 (orange). C) The A/T conformation requires a ~30° bend in the tRNA body (green) compared to a canonical tRNA (grey). This bend is achieved through two isolated regions of distortion, first in the anticodon stem and the second in the D-stem, where both the A9C and G24A mutations are located.

Figure 2

Comparison of cognate and near-cognate structures in the decoding center. A) Unbiased F_o - F_c electron density (displayed at 1.3σ within 2 Å of EF-Tu) is shown for residues in the wobble position of the codon-anticodon helix. B) Binding of a near-cognate tRNA (A9C on UGA: orange, G24A on UGA: red), compared to a cognate tRNA (G24A on UGG: yellow, tRNA^Trp on UGG: green), results in a shift in both the tRNA anticodon and mRNA codon. C) The distortion in the anticodon caused by this mismatch is propagated three residues, resulting in a shift in the tRNA backbone for the G24A on UGA when compared to the G24A or tRNA^Trp on UGG. D) However this distortion does not continue past residue 31; by residue 28 the backbone of the G24A on UGA has converged with that of the G24A on UGG.

Figure 3

Miscoding by the G24A and A9C Trp-tRNA^Trp. A) Mutation of G24A (red) facilitates formation of a hydrogen bonding interaction that is not possible in native tRNA^Trp (green) between the exocylic amine of A24 and O6 of G44. This interaction is facilitated by a small shift in the RNA backbone between residues 20 to 30 and a commensurate movement between residues 9-16, B) The interaction between residues 24 and 44 in the G24A tRNA^Trp is only possible because of a base pair between residues A26-U45 in tRNA^Trp that dictates the backbone conformation in this region. In contrast, tRNA^Thr14(purple) contains two purines, G26 and G45, at these positions, which push the tRNA backbone apart and separate residues G24 and A44. That the A26-U45 base pair is not conserved even throughout bacterial tRNA^Trp further illustrates that evolution has fine-tuned each tRNA to find a unique solution to tRNA bending during decoding. (C) In native tRNA^Trp (shown in green) a base triple forms between residues 9:12:23, which is at the junction of three separate strands of the tRNA. Mutation of residue 9 to a cytosine (shown in orange) weakens both the packing and hydrogen-bonding of this base-triple, which could result in higher flexibility of the tRNA body and explain its ability to miscode.