FIGURE SUMMARY
Title

A chiral selectivity relaxed paralog of DTD for proofreading tRNA mischarging in Animalia

Authors
Kuncha, S.K., Mazeed, M., Singh, R., Kattula, B., Routh, S.B., Sankaranarayanan, R.
Source
Full text @ Nat. Commun.
Fig. 1

ATD is a variant of DTD. a Multiple sequence alignment showing similar but distinct and characteristic sequence motifs in DTD and ATD (motifs 1 and 2). The highly conserved arginine in DTD (Arg7, EcDTD) is indicated by a star above, whereas the invariant arginine in ATD (Arg151, MmATD) is highlighted by a star below. b Phylogenetic classification of DTD and ATD showing their grouping into two separate categories. c Crystal structure of MmATD homodimer (blue) superimposed on that of PfDTD homodimer (cyan; PDB id: 4NBI)
Fig. 2

ATD has similar active site features as compared to DTD’s. a Crystal structures of PfDTD (PDB id: 4NBI) and MmATD showing that motifs 1 and 2 form the active site at the dimeric interface in both. b Structural superposition of MmATD on PfDTD displaying the cross-subunit Gly-Pro motif in both, i.e., the motif from one monomer inserted into the active site of the other monomer. Residues from the dimeric counterpart are indicated by *
Fig. 3

ATD has a Gly-transPro motif in the active site, unlike a Gly-cisPro motif in DTD. a Comparison between Gly-transPro motif in MmATD and Gly-cisPro motif in PfDTD (PDB id: 4NBI) after structural superposition of the two proteins. b The comparison shown in a depicted from a different angle, highlighting the opposite orientation of Gly-Pro carbonyl oxygens of the two proteins. c Ramachandran plot of glycine and proline residues of the Gly-Pro motif of all the available crystal structures of DTD (blue) and ATD (red), highlighting the change of ~180° in the ψ torsion angle. d Interaction of the side chain of Arg7 with the Gly-cisPro motif of the same monomer in PfDTD (PDB id: 4NBI), and of the side chain of Arg151 with the Gly-transPro motif of the dimeric counterpart in MmATD. Residues from the dimeric counterpart are indicated by *
Fig. 4

ATD displays relaxation of substrate chiral specificity. a–d Deacylation of d-Tyr-tRNATyr, l-Tyr-tRNATyr, Gly-tRNAGly and l-Ala-tRNAAla by different concentrations of MmATD. Aminoacyl-tRNAs were used at 200 nM final concentration. Error bars denote one standard deviation from the mean of three independent readings
Fig. 5

Proofreading of L-Ala-tRNAThr(G4•U69) by ATD is conserved across organisms. Deacylation of l-Ala-tRNAThr(G4•U69) and l-Thr-tRNAThr(G4•U69) by different concentrations of a MmATD, b HsATD, c GgATD, and d DrATD. Aminoacyl-tRNAs were used at 200 nM final concentration. Er`ror bars denote one standard deviation from the mean of three independent readings
Fig. 6

EF-Tu confers protection on L-Thr-tRNAThr(G4•U69) against ATD. Deacylation of l-Ala-tRNAThr(G4•U69) by different concentrations of MmATD in the presence of a unactivated EF-Tu, and b activated EF-Tu. Deacylation of l-Thr-tRNAThr(G4•U69) by different concentrations of MmATD in the presence of c unactivated EF-Tu, and d activated EF-Tu. Aminoacyl-tRNAs and EF-Tu were used at 200 nM and 2 µM final concentration, respectively. Error bars denote one standard deviation from the mean of three independent readings
Fig. 7

Enrichment of tRNAThr(G4•U69) genes and presence of ATD show strict association. a Distribution of AlaRSND, tRNAThr(G4•U69) genes, and ATD in different domains of life. tRNA gene sequences of Cnidaria and Mollusca are not available in the database. b Bar graph (logarithmic scale) depicting genome size, total number of tRNAThr genes, and number of tRNAThr(G4•U69) genes occurring in representative organisms belonging to all the three domains of life. Inset showing the number of total tRNAThr genes and tRNAThr(G4•U69) genes in normal scale; genome size has not been shown for the sake of clarity. Presence of ATD is highlighted in light blue box. Data for occurrence of AlaRSND and tRNAThr(G4•U69) genes have been taken from reference 53. c Bar graph showing the number of organisms containing (G4•U69)-harboring tRNA genes which code for tRNAs specific for various proteinogenic amino acids
Fig. 8

ATD is a unique and dedicated proofreading factor that rectifies a critical tRNA selection error. Model for mis-selection and consequent misacylation of tRNAThr(G4•U69) with l-alanine by AlaRSND, and its subsequent proofreading by ATD. Cognate and non-cognate tRNAs (clover leaf model) are colored in green and red, respectively. Likewise, cognate and non-cognate amino acids (circle) are rendered in green and red, respectively
Acknowledgments
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