Evolutionary Gain of Alanine Mischarging to Noncognate tRNAs with a G4:U69 Base Pair

Abstract

Fidelity of translation, which is predominately dictated by the accuracy of aminoacyl-tRNA synthetases in pairing amino acids with correct tRNAs, is of central importance in biology. Yet, deliberate modifications of translational fidelity can be beneficial. Here we found human and not E. coli AlaRS has an intrinsic capacity for mispairing alanine onto nonalanyl-tRNAs including tRNA^Cys. Consistently, a cysteine-to-alanine substitution was found in a reporter protein expressed in human cells. All human AlaRS-mischarged tRNAs have a G4:U69 base pair in the acceptor stem. The base pair is required for the mischarging. By solving the crystal structure of human AlaRS and comparing it to that of E. coli AlaRS, we identified a key sequence divergence between eukaryotes and bacteria that influences mischarging. Thus, the expanded tRNA specificity of AlaRS appears to be an evolutionary gain-of-function to provide posttranscriptional alanine substitutions in eukaryotic proteins for potential regulations.

Figure 1. Human AlaRS and not TyrRS and GlyRS mischarge non-cognate human tRNAs

(a) Cloverleaf secondary structure of human tRNA^Ala, tRNA^Tyr, and tRNA^Gly. The identity elements are highlighted with blue shadow for tRNA^Ala, and with purple shadows for tRNA^Tyr and tRNA^Gly. The anticodon nucleotides are in red. (b) Microarray showing misacylation of non-cognate tRNAs by human AlaRS after 5 or 30 min of reaction with total human tRNAs and [³H]-alanine (black, Ala-tRNA^Ala; burgundy, Ala-tRNA^Cys; blue, Ala-tRNA^Thr; green, Ala-tRNA^mAsp). (c) Microarray analysis after blocking with a large excess of all tRNA^Ala probes to exclude the potential signal from ³H-Ala-tRNA^Ala cross-hybridizing to tRNA^Cys, tRNA^Thr or mt-tRNA^Asp probes. The relative remaining signal after crosshybridization is quantified separately for tRNA^Ala, tRNA^Cys, tRNA^Thr and mt-tRNA^Asp. (d, e, f) No mischarging activity is detected with human TyrRS (d), GlyRS (e), and GlyRS without the metazoan-specific WHEP domain (f).

Figure 2. G4:U69 base pair in tRNA is responsible for human AlaRS mischarging

(a) G:U base pair location indicated on the cloverleaf secondary structure of different tRNAs. (b) In vitro aminoacylation assay showing that human AlaRS has mischarging activity with G4:U69-containing tRNAs. Assays were carried out with 200 nM AlaRS and 4 μM cognate tRNA^Ala or 25 μM non-cognate tRNAs. Inset shows the zoom-in view of the mischarging activity of AlaRS with G4:U69-tRNA^Thr. Error bars indicate standard deviations. (c) Mutational analysis on tRNA^Cys in both gain-of-function and loss-of-function manners confirming that the mischarging activity of AlaRS is dependent on the G4:U69 base pair. (d) Mass spectrometry analysis of a flag-CaMKII protein expressed and purified from HEK293T cells identified a Cys-to-Ala substitution.

Figure 3. E. coli AlaRS does not mischarge non-cognate human tRNAs

(a) In vitro aminoacylation assay showing that E.coli AlaRS has no mischarging activity towards non-cognate tRNAs. Assays were carried out with 200 nM E.coli AlaRS and 4 μM cognate human tRNA^Ala or 25 μM non-cognate human tRNAs. (b) Conservation analysis of AlaRS sequences across bacteria, archaea, and eukaryotes. (c,d) Specific recognitions of the G3:U70 identity element in tRNA^Ala by AlaRS. The recognition is adapted from the crystal structure of A. fulgidus AlaRS and tRNA^Ala complex (PDB 3WQY) (CD, catalytic domain; TBD, tRNA binding domain; ED, editing domain; C-Ala, C-Ala domain).

Figure 4. Crystal structure of human AlaRS

(a) The co-crystal structure of human AlaRS^N455 and AlaSA. Catalytic domain is colored in orange and tRNA binding domain is colored in blue. The three conserved sequence motifs (Motif I, II, and III) in the catalytic domain are highlighted with different colors. AlaSA is bound in the active site within the catalytic domain. (b) Superimposition of the crystal structure of human AlaRS^N455 and of E. coli AlaRS (PDB 3HXU). Human AlaRS⁴⁵⁵ is colored as indicated above, while E. coli AlaRS is colored in pink. (c) A zoom-in view showing human AlaRS lost the Gln-Asp interaction found in E. coli AlaRS. (d) In vitro aminoacylation assay showing Gln-to-Ala (Q432A) substitution in E. coli AlaRS is unable to mischarge human G4:U69-tRNA^Cys. Assays were carried out with 200 nM of WT or Q432A AlaRS, and 2.5 μM tRNA^Ala or 25 μM G4:U69-tRNA^Cys. (e) Ala-to-Gln (A448Q) substitution reduces the mischarging activity of human AlaRS towards G4:U69-tRNA^Cys. The aminoacylation assays were carried out with 200 nM of WT or A448Q AlaRS, and 2 μM tRNA^Ala or 10 μM G4:U69-tRNA^Cys.

Figure 5. Distribution of G4:U69-containing tRNA genes in plants, vertebrates and mammals

Only tRNA genes with tRNAScan score of >50 are included here. These tRNAs have a high likelihood to form the cloverleaf secondary structure.