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

Abstract

10D-aminoacyl-tRNA deacylase (DTD) acts on achiral glycine, in addition to D-amino acids, 11 attached to tRNA. We have recently shown that this activity enables DTD to clear non-cognate 12 Gly-tRNA Ala with 1000-fold higher efficiency than its activity on Gly-tRNA Gly , indicating 13 tRNA-based modulation of DTD (Pawar et al., 2017). Here, we show that tRNA's 14 discriminator base predominantly accounts for this activity difference and is the key to 15 selection by DTD. Accordingly, the uracil discriminator base, serving as a negative 16 determinant, prevents Gly-tRNA Gly misediting by DTD and this protection is augmented by 17 EF-Tu. Intriguingly, eukaryotic DTD has inverted discriminator base specificity and uses only 18 G3•U70 for tRNA Gly/Ala discrimination. Moreover, DTD prevents alanine-to-glycine 19 misincorporation in proteins rather than only recycling mischarged tRNA Ala . Overall, the study 20 reveals the unique co-evolution of DTD and discriminator base, "reciprocally" in Bacteria and 21 Eukarya, and suggests DTD's strong selection pressure on bacterial tRNA Gly s to retain a 22 pyrimidine discriminator code. 23 24 Quality control during translation of the genetic code involves multiple stages and a multitude 25 of proofreading factors (Guo and Schimmel, 2012; Ibba and Söll, 2000; Ogle and 26 Ramakrishnan, 2005). A compromise in editing leads to serious pathologies including 27 neurodegeneration in mouse, and even cell death (Bacher et al., 2005; Bullwinkle et al., 2014; 28 Karkhanis et al., 2007; Korencic et al., 2004; Lee et al., 2006; Liu et al., 2014; Lu et al., 29 2014; Moghal et al., 2016; Nangle et al., 2002; Roy et al., 2004). Among these proofreading 30 factors, D-aminoacyl-tRNA deacylase (DTD) is the one that specifically decouples wrongly 31 acylated D-amino acids from tRNAs (Calendar and Berg, 1967; Soutourina et al., 1999, 32 2000). Our studies have shown that DTD is an RNA-based catalyst that uses an invariant Gly-33 cisPro motif as a "chiral selectivity filter" to achieve substrate chiral specificity only through 34 rejection of L-amino acid from the active site, thereby leading to Gly-tRNA Gly misediting 35 (Ahmad et al., 2013; Routh et al., 2016; Routh and Sankaranarayanan, 2017). Recently, 36we have also shown that DTD's activity on achiral glycine helps in clearing Gly-tRNA Ala , a 37 misaminoacylation product of alanyl-tRNA synthetase (AlaRS), thus resolving a long-standing 38 question in translational quality control (Pawar et al., 2017). 39 As DTD acts on multiple tRNAs charged with D-amino acids or glycine, tRNA's role 40 in modulating DTD's activity was previously thought to be inconsequential (Calendar and 41 Berg, 1967). However, our recent work has shown that DTD's activity on Gly-tRNA Ala is about 42 1000-fold higher than on Gly-tRNA Gly , clearly demonstrating the profound effect of tRNA 43 elements on DTD and suggesting an underlying tRNA code for DTD's action. G3•U70, the 44 universal tRNA Ala -specific determinant for AlaRS, is also a determinant for DTD. Ho...