Switching a conflicted bacterial DTD-tRNA code is essential for the emergence of mitochondria

Optimization of DTD-tRNA code and mito-tRNA(Gly) discriminator base is important for emergence of mitochondria.

Development of Assay Systems for Amber Codon Decoding at the Steps of Initiation and Elongation in Mycobacteria

ABSTRACTGenetic analysis of the mechanism of protein synthesis in Gram-positive bacteria has remained largely unexplored because of the unavailability of appropriatein vivoassay systems. We developed chloramphenicol acetyltransferase (CAT)-basedin vivoreporter systems to study translation initiation and elongation inMycobacterium smegmatis. The CAT reporters utilize specific decoding of amber codons by mutant initiator tRNA (i-tRNA,metU) molecules containing a CUA anticodon (metUCUA). The assay systems allow structure-function analyses of tRNAs without interfering with the cellular protein synthesis and function with or without the expression of heterologous GlnRS fromEscherichia coli. We show that despite their naturally occurring slow-growth phenotypes, the step of i-tRNA formylation is vital in translation initiation in mycobacteria and that formylation-deficient i-tRNA mutants (metUCUA/A1,metUCUA/G72, andmetUCUA/G72G73) with a Watson-Crick base pair at the 1·72 position participate in elongation. In the absence of heterologous GlnRS expression, the mutant tRNAs are predominantly aminoacylated (glutamylated) by nondiscriminating GluRS. Acid urea gels show complete transamidation of the glutamylatedmetUCUA/G72G73tRNA to its glutaminylated form (by GatCAB) inM. smegmatis. In contrast, the glutamylatedmetUCUA/G72tRNA did not show a detectable level of transamidation. Interestingly, themetUCUA/A1mutant showed an intermediate activity of transamidation and accumulated in both glutamylated and glutaminylated forms. These observations suggest important roles for the discriminator base position and/or a weak Watson-Crick base pair at 1·72 forin vivorecognition of the glutamylated tRNAs byM. smegmatisGatCAB.IMPORTANCEGenetic analysis of the translational apparatus in Gram-positive bacteria has remained largely unexplored because of the unavailability of appropriatein vivoassay systems. We developed chloramphenicol acetyltransferase (CAT)-based reporters which utilize specific decoding of amber codons by mutant tRNAs at the steps of initiation and/or elongation to allow structure-function analysis of the translational machinery. We show that formylation of the initiator tRNA (i-tRNA) is crucial even for slow-growing bacteria and that i-tRNA mutants with a CUA anticodon are aminoacylated by nondiscriminating GluRS. The discriminator base position, and/or a weak Watson-Crick base pair at the top of the acceptor stem, provides important determinants for transamidation of the i-tRNA-attached Glu to Gln by the mycobacterial GatCAB.

A discriminator code–based DTD surveillance ensures faithful glycine delivery for protein biosynthesis in bacteria

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.

A discriminator code-based DTD surveillance ensures faithful glycine delivery for protein biosynthesis in bacteria

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, “reciprocally” in Bacteria and Eukarya, and suggests DTD’s strong selection pressure on bacterial tRNAGlys to retain a pyrimidine discriminator code.