Superwobbling facilitates translation with reduced tRNA sets

Abstract

Some bacterial and most organelle genomes do not encode the full set of 32 tRNA species required to read all codons according to Crick's wobble rules. 'Superwobble', in which a tRNA species with an unmodified U in the wobble position reads all four nucleotides in the third codon position, represents one possible mechanism for how a reduced tRNA set could still suffice. We have tested the superwobble hypothesis by producing knockout mutants for the pair of plastid glycine tRNA genes. Here we show that, whereas the tRNA gene with U in the wobble position is essential, the gene with G in this position is nonessential, demonstrating that the U-containing anticodon can indeed read all four glycine triplets. We also show that the price for superwobbling is a reduced translational efficiency, which explains why most organisms prefer pairs of isoaccepting tRNAs over the superwobbling mechanism.

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Figure 1: Construction of transformation vectors for disruption of the two plastid tRNAGly genes.
Figure 2: Mutant phenotypes of plastid transformants generated with the knockout constructs for the two glycine tRNAs.
Figure 3: Seed assays to confirm heteroplasmy of ΔtrnG-UCC plants and homoplasmy of ΔtrnG-GCC plants.
Figure 4: Analysis of plastid gene expression in tRNAGly mutants.
Figure 5: Absence of tRNA import into chloroplasts of the ΔtrnG-GCC mutant.

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Acknowledgements

We thank the MPI-MP Green Team for plant care and cultivation and S. Ruf for helpful discussion and advice. We are grateful to J. Pieritz and J. Kehr for help with mass spectrometric protein sequencing. M.R. is the recipient of a fellowship from the Deutscher Akademischer Austauschdienst (DAAD, Germany) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil). This research was financed by the Max Planck Society and a grant from the Deutsche Forschungsgemeinschaft.

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Correspondence to Ralph Bock.

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Supplementary Fig. 1, Table 1 & Methods (PDF 203 kb)

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Rogalski, M., Karcher, D. & Bock, R. Superwobbling facilitates translation with reduced tRNA sets. Nat Struct Mol Biol 15, 192–198 (2008). https://doi.org/10.1038/nsmb.1370

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