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.

Conserved residues in yeast initiator tRNA calibrate initiation accuracy by regulating preinitiation complex stability at the start codon

“…Consistent with this, mutations that reduce t 6 A37 formation in yeast impair recognition of AUG codons (Lin et al 2009;Daugeron et al 2011;Srinivasan et al 2011) and can increase the ratio of GUG to AUG initiation . To eliminate the possibility that G31:C39 substitutions confer Sui À phenotypes by impairing t 6 A37 formation, we purified wild-type and G31A:C39U mutant tRNA i , digested them with nuclease P1, and resolved the nucleoside products by high-performance liquid chromatography (HPLC).…”

Protein folding and tRNA biology

“…In Archaea and Eukarya, this process involves a universal protein (Sua5) and the complex KEOPS/EKC (Srinivasan et al 2011). KEOPS/ EKC was early associated with different cellular processes: transcription regulation, telomere homeostasis, genome instability, chromosome segregation, and metabolic regulation (Srinivasan et al 2011). The t6A modification has been associated with cell growth regulation (Rojas-Benitez et al 2015) and the levels of t6A-modified tRNA strongly influence TORC1 activity (Rojas-Benitez et al 2015).…”

“…In Archaea and Eukarya, this process involves a universal protein (Sua5) and the complex KEOPS/EKC (Srinivasan et al 2011). KEOPS/ EKC was early associated with different cellular processes: transcription regulation, telomere homeostasis, genome instability, chromosome segregation, and metabolic regulation (Srinivasan et al 2011).…”

Transfer RNA Post-Transcriptional Processing, Turnover, and Subcellular Dynamics in the YeastSaccharomyces cerevisiae

“…Similarly, mutations of genes encoding proteins of the KEOPS complex cause growth defects and telomere shortening. However, the KEOPS complex along with Sua5 is required for ct 6 A modification, which functions in decoding ANN codons, including appropriate initiation at AUG (Daugeron et al 2011;El Yacoubi et al 2012;reviewed in Hinnebusch 2011;Srinivasan et al 2011).…”