The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A

Abstract

The highly conserved Kinase, Endopeptidase and Other Proteins of small Size (KEOPS)/Endopeptidase-like and Kinase associated to transcribed Chromatin (EKC) protein complex has been implicated in transcription, telomere maintenance and chromosome segregation, but its exact function remains unknown. The complex consists of five proteins, Kinase-Associated Endopeptidase (Kae1), a highly conserved protein present in bacteria, archaea and eukaryotes, a kinase (Bud32) and three additional small polypeptides. We showed that the complex is required for a universal tRNA modification, threonyl carbamoyl adenosine (t6A), found in all tRNAs that pair with ANN codons in mRNA. We also showed that the bacterial ortholog of Kae1, YgjD, is required for t6A modification of Escherichia coli tRNAs. The ATPase activity of Kae1 and the kinase activity of Bud32 are required for the modification. The yeast protein Sua5 has been reported previously to be required for t6A synthesis. Using yeast extracts, we established an in vitro system for the synthesis of t6A that requires Sua5, Kae1, threonine, bicarbonate and ATP. It remains to be determined whether all reported defects of KEOPS/EKC mutants can be attributed to the lack of t6A, or whether the complex has multiple functions.

Figure 1

ClustalW alignment of eukaryotic Kae1 orthologs. The alignment of the amino acid sequences was generated using Vector NTI software (Invitrogen). Identical residues are shaded in yellow and conservative substitutions are shaded in green. Dm, Drosophila melanogaster; h, Homo sapiens; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe.

Figure 2

Primer extension analysis of tRNA Ile ^(AAU) and Val ^(UAC). tRNAs isolated from wild type (WT), kae1Δ and sua5Δ mutant strains were subjected to primer extension analysis as described in Materials and methods. End-labeled primers specific to either tRNA Ile ^(AAU) or Val ^(UAC) were used for the analysis. Cloverleaf structures of the Ile and Val tRNAs are shown. The specific primers used are complementary to the sequence shown in bold face in the cloverleaf structures. The position of the t6A-modified base (A37) in the Ile tRNA, marked by horizontal arrows, was confirmed by running length standards and sequence ladders.

Figure 3

(A) LC-MS/MS spectrum of t6A. Based on the fragmentation of an adenosine standard, t6A was analysed by LC-MS/MS in selected reaction monitoring (SRM) mode (m/z 413.2 → m/z 280.9). (B) t6A content of tRNA isolated from the indicated strains. tRNA isolated from wild type (WT), kae1Δ, sua5Δ, qri7Δ and kae1Δ qri7Δ mutant strains was hydrolysed to nucleosides as described in Materials and methods. The t6A content of each strain, normalized to the respective adenosine content, is plotted as the % t6A/A and the numerical values are also shown below the graph. (C) t6A content of an E. coli strain depleted of YgjD. An E. coli strain with the natural ygjD promoter replaced by the inducible arabinose promoter was grown with (YgjD⁺) and without arabinose (YgjD⁻). tRNA was isolated from the two cultures and processed for LC-MS/MS analysis as above. The % t6A/A for each condition is plotted and the numerical values are also shown below the graph.

Figure 4

Primer extension analysis of tRNA Ile ^(AAU) isolated from KEOPS/EKC mutants. tRNAs isolated from wild type (WT), pcc1Δ, kae1Δ, bud32Δ and cgi121Δ mutant strains were subjected to primer extension analysis as in Figure 1. The cloverleaf structure of the Ile tRNA is shown. The specific primer used for the analysis is complementary to the sequence shown in bold face in the cloverleaf structure. The position of the t6A-modified base (A37) in the Ile tRNA is marked by horizontal arrows.

Figure 5

Kae1 ATPase activity and Bud32 kinase activity are required for t6A formation. (A) LC-MS/MS analysis of tRNA nucleosides from kae1 mutants. A kae1Δ strain was transformed with a low copy CEN plasmid containing the wild-type KAE1 gene (WT), a mutant of the ATP-binding site (kae1^H141,145A) or the empty vector (kae1Δ). Ten-fold serial dilutions of the respective cultures were spotted onto a selective medium and assessed for growth at 30°C. tRNA isolated from the three strains was processed for LC-MS/MS quantification of t6A and A as described in Materials and methods. The t6A content of each sample, normalized to the respective adenosine content, is plotted as the % t6A/A and the numerical values are also shown below the graph. (B) A similar analysis was done with bud32 mutants. A bud32Δ strain was transformed with a low copy CEN plasmid containing the wild-type BUD32 gene (WT), a catalytic site mutant (bud32^D161A) or the empty vector (bud32Δ). Ten-fold serial dilutions of the respective cultures were spotted onto a selective medium and assessed for growth at 30°C. The t6A content of each sample, normalized to the respective adenosine content, is plotted as the % t6A/A and the numerical values are also shown below the graph.

Figure 6

(A) In vitro t6A biosynthesis requires bicarbonate and ATP. The top panel shows the retention time (RT=12.5 min) and m/z value (413.2) of t6A as measured by LC-MS/MS. The three panels below that show the results found when t6A-deficient tRNA was incubated with wild-type cell extract and heavy threonine (one ¹⁵N and four ¹³C atoms), in presence of bicarbonate and ATP (complete), lacking bicarbonate (−HCO₃⁻), or lacking ATP (−ATP). The t6A heavy isotope-labeled reaction product was confirmed by LC-MS/MS in SRM mode (m/z 418.6 → m/z 286.0). (B) In vitro t6A biosynthesis requires Kae1 and Sua5. t6A-deficient tRNA was incubated with cell extracts prepared from wild type, sua5Δ or kae1Δ strains in the presence of heavy threonine, bicarbonate and ATP. The reaction products were processed and analysed as in (A). The mass spectrum of the nucleotide component with a RT of 12.5 min is shown.