A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification

Abstract

The YgjD/Kae1 family (COG0533) has been on the top-10 list of universally conserved proteins of unknown function for over 5 years. It has been linked to DNA maintenance in bacteria and mitochondria and transcription regulation and telomere homeostasis in eukaryotes, but its actual function has never been found. Based on a comparative genomic and structural analysis, we predicted this family was involved in the biosynthesis of N(6)-threonylcarbamoyl adenosine, a universal modification found at position 37 of tRNAs decoding ANN codons. This was confirmed as a yeast mutant lacking Kae1 is devoid of t(6)A. t(6)A(-) strains were also used to reveal that t(6)A has a critical role in initiation codon restriction to AUG and in restricting frameshifting at tandem ANN codons. We also showed that YaeZ, a YgjD paralog, is required for YgjD function in vivo in bacteria. This work lays the foundation for understanding the pleiotropic role of this universal protein family.

Figure 1

Domain organization of the YrdC, YgjD, and Sua5 domain containing proteins. (A) Each protein is shown as a bar, the length of which is approximately proportional to the number of residues; the YrdC-like domains are shaded dark grey, and the YgjD-like domains are shaded light grey. Positions of the conserved motifs in the two domain families are marked by numbered boxes. In YeaZ, only the N-terminal region has homology to the YgjD domains, and there are no conserved motifs identifiable in the rest of its chain. (B) Sequence signatures of the conserved motifs (marked on panel A). All numbering is based on the E. coli YgjD protein (NP_417536.1).

Figure 2

LC–MS/MS analysis of yeast tRNA extracted from different strains carrying homologs of the Kae1/YgjD/Qri7 family. Extracted ion chromatograms for 413 m/z are shown for each strain under the UV traces. (A) t⁶A profiles linking the disappearance of the t⁶A peak to the deletion of KAE1. WT (BY4741) (left panels), kae1Δ (middle panels), and kae1Δ carrying KAE1 in trans (right panels). (B) t⁶A profiles of kae1Δ expressing the homologue from E. coli YgjD (pYESygjD) (left panels) or the yeast mitochondria located QRI7 (pYESQRI7) (right panels). Positive and negative controls were run as presented in (A). (C) Growth phenotypes of yeast strains lacking a functional KAE1 and transformed with pYES, pYESKAE1_Sc, or pYESQRI7_Sc. The parent BY4741 was transformed with pYES and used as reference. Each growth curve presented here is an average of 10 independent growth curves. Error bars represent s.d. The growth conditions are described in Supplementary data.

Figure 3

Role of t⁶A in the frequency +1 and –1 frameshifts. (A) Sequence of the frameshift-inducing targets. (B) Frameshifting frequencies for target sequence BLV, Prrvs, and EST3. Measurements are average of five independent measurements using a dual reporter construct with the target sequence inserted between the lacZ and firefly luciferase ORFs and a control constructs containing the corresponding in-frame sequence inserted in between. Efficiency of frameshifting, expressed as percentage, was calculated by dividing the firefly luciferase/β-galactosidase ratio obtained for each test construct by the same ratio obtained for the corresponding in-frame control construct. Errors bars represent s.d.

Figure 4

Lack of t⁶A in yeast affects initiation codon selection in vivo. The initiation efficiency is defined as the ratio of the firefly luciferase activity initiated on AUG or GUG codon to the one of the renilla luciferase activity initiated on AUG codon. The relative activities presented correspond to the ratios for the t⁶A⁻ strains (deletion mutants sua5Δ and kae1Δ) normalized by the ratio obtained for wild-type strain BY4741 with the firefly luciferase and the renilla luciferase both initiated on AUG. All the measurements were the average of five independent extractions. Errors bars represent standard deviations.

Figure 5

Genetic complementation of the ygjD essentiality phenotype in E. coli carrying the ygjD gene under the control of the P_TET promoter. (A) Phenotype of the P_TET:ygjD strain transformed with control plasmid pBAD24 or plasmid pBADygjD_Ec (pBAD24 carrying ygjD_Ec) grown under inducing conditions (aTc and ara) or repressing condition (glu). (B) Lack of complementation of the essentiality phenotype of ygjD_Ec by the yeast homologs KAE1 and QRI7 (lacking the sequence of the 30 AA targeting signal) and the archaeal homologue for M. maripaludis kae1-prpk. Cells grew in the presence of aTc allowing the expression of the chromosomal ygjD_Ecallele but failed to grow in the absence of aTc and the presence of arabinose allowing the expression of the plasmid borne alleles.

Figure 6

The B. subtilis ygjD homologue functionally replaces the E. coli homologue only when coexpressed with the yeaZ gene from B. subtilis. (A) The physical clustering deduced from analysing the genetic context of both ygjD and yeaZ suggest that these two genes function in the same pathway. (B) Model of YgjD–YeaZ heterodimer. The N-terminal domain of the YgjD homologue Kae1 (orange, PDB 2IVP) is superimposed on the related domain of one subunit of the YeaZ homodimer (silver; PDB 1OKJ), resulting in the conserved interface with the other YeaZ subunit (blue). (C) Genetic complementation of the essentiality of E. coli ygjD by coexpression of the B. subtilis ygjD and YeaZ in an operonic structure. The P_TET:ygjD strain was transformed with plasmid pBADygjD_Bs carrying the yeaZ_Bs in an operonic structure (pBADygjD_BsyeaZ_Bs) or in the reverse orientation (pBADygjD_BsoppyeaZ_Bs) and tested for growth under inducing conditions (aTc and ara) or repressing condition (glu). pBADygjD_Ec, pBADygjD_EcyeaZ_Ec, and pBAD24 were used as positive and negative controls, respectively.

Figure 7

Fitting ligands into the putative active sites of the YrdC/Sua5 and YgjD/Kae1 families. (A) The product carbamoylthreonine (stick, yellow carbons) is in the active site of Kae1 (2IVP). The carbamoyl group contacts the bound metal ion (magenta sphere). Those conserved residues not in the ATP or metal ion-binding sites are shown with opaque spheres, cyan carbons). (B) ATP (stick, green carbons) and the substrate carbamoylthreonine (stick, yellow carbons) are in the active site of the YrdC domain of Sua5 (PDB id: 2EQA). The AMP group of the ATP molecule is taken from the Sua5 structure; the pyrophospate group is docked with the KRSN tetrad (opaque spheres, magenta carbons). Carbamoylthreonine sits in the pocket of conserved residues with carbamoyl group contacting the α-phosphate group. (C) Proposed t⁶A biosynthesis pathway.