The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA

Abstract

Threonylcarbamoyladenosine (t6A) is a universal modification found at position 37 of ANN decoding tRNAs, which imparts a unique structure to the anticodon loop enhancing its binding to ribosomes in vitro. Using a combination of bioinformatic, genetic, structural and biochemical approaches, the universal protein family YrdC/Sua5 (COG0009) was shown to be involved in the biosynthesis of this hypermodified base. Contradictory reports on the essentiality of both the yrdC wild-type gene of Escherichia coli and the SUA5 wild-type gene of Saccharomyces cerevisiae led us to reconstruct null alleles for both genes and prove that yrdC is essential in E. coli, whereas SUA5 is dispensable in yeast but results in severe growth phenotypes. Structural and biochemical analyses revealed that the E. coli YrdC protein binds ATP and preferentially binds RNAThr lacking only the t6A modification. This work lays the foundation for elucidating the function of a protein family found in every sequenced genome to date and understanding the role of t6A in vivo.

The Levels of a Universally Conserved tRNA Modification Regulate Cell Growth

“…mined that this enzyme is necessary for cell autonomous growth in Drosophila, as we previously showed for Tcs5 (Prpk) (23). Metazoans are composed of different cell types, therefore making the phenotype observed in tcs3 mutants even more complex than the ones observed in yeast and other unicellular organisms (15,16,18,21,50,51). However, in every case analyzed, the deficiency of these enzymes strongly impairs cellular growth and proliferation.…”

“…tRNA Extraction and Detection by HPLC-For yeast and Drosophila tRNA extraction the method by El Yacoubi et al (16) was used. HPLC analysis was performed as described in Ref.…”

“…In addition, considering the particular limiting nature of the initiator tRNA for cell and animal growth (5,7) and as this tRNA is modified by t 6 A, we were prompted to investigate the impact of t 6 A in whole animal context using Drosophila. All previous studies on t 6 A function and synthesis have been performed in unicellular organisms or cells in culture (15)(16)(17)(18)(19)(20)(21). In this study, we address for the first time the role of t 6 A in a metazoan.…”

“…It stabilizes codon-anticodon interaction (13), suggesting that t 6 A might have a role regulating protein synthesis initiation. The modification was discovered over 40 years ago (14), but the enzymes involved in t 6 A synthesis were only identified and characterized in all domains of life in the last few years (15)(16)(17)(18)(19)(20)(21) and recently renamed (22). In yeast, components of the threonyl-carbamoyl transferase complex (TCTC, previously named KEOPS/EKC (kinase, endopeptidase and other proteins of small size/endopeptidase-like and kinase associated to transcribed chromatin) are required for t 6 A synthesis.…”

“…Absence of t 6 A leads to an increase in ϩ1 and Ϫ1 frameshifts and increases non-AUG start sites (16), which could explain its requirement for growth in eukaryotes. In addition, considering the particular limiting nature of the initiator tRNA for cell and animal growth (5,7) and as this tRNA is modified by t 6 A, we were prompted to investigate the impact of t 6 A in whole animal context using Drosophila.…”