Switching a conflicted bacterial DTD-tRNA code is essential for the emergence of mitochondria.
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ABSTRACT:
SUBMITTER: Gogoi J
PROVIDER: S-EPMC8754408 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:D-aminoacyl-tRNA deacylase (DTD) acts on achiral glycine, in addition to D-amino acids, attached to tRNA. We have recently shown that this activity enables DTD to clear non-cognate Gly-tRNAAla with 1000-fold higher efficiency than its activity on Gly-tRNAGly, indicating tRNA-based modulation of DTD (Pawar et al., 2017). Here, we show that tRNA's discriminator base predominantly accounts for this activity difference and is the key to selection by DTD. Accordingly, the uracil discriminator base, serving as a negative determinant, prevents Gly-tRNAGly misediting by DTD and this protection is augmented by EF-Tu. Intriguingly, eukaryotic DTD has inverted discriminator base specificity and uses only G3•U70 for tRNAGly/Ala discrimination. Moreover, DTD prevents alanine-to-glycine misincorporation in proteins rather than only recycling mischarged tRNAAla. Overall, the study reveals the unique co-evolution of DTD and discriminator base, and suggests DTD's strong selection pressure on bacterial tRNAGlys to retain a pyrimidine discriminator code.
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Project description:The bacteria-derived tyrosyl-tRNA synthetase (TyrRS)/tRNA pair was first used for unnatural amino acid (Uaa) mutagenesis in eukaryotic cells over 15 years ago. It provides an ideal platform to genetically encode numerous useful Uaas in eukaryotes. However, this pair has been engineered to charge only a small collection of Uaas to date. Development of Uaa-selective variants of this pair has been limited by technical challenges associated with a yeast-based directed evolution platform, which is currently required to alter its substrate specificity. Here we overcome this limitation by enabling its directed evolution in an engineered strain of E. coli (ATMY), where the endogenous TyrRS/tRNA pair has been functionally replaced with an archaeal counterpart. The facile E. coli-based selection system enabled rapid engineering of this pair to develop variants that selectively incorporate various Uaas, including p-boronophenylalanine, into proteins expressed in mammalian cells as well as in the ATMY strain of E. coli.
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