Structure-function analysis of Sua5 protein reveals novel functional motifs required for the biosynthesis of the universal t 6 A tRNA modification

RNA. 2018 Jul;24(7):926-938. doi: 10.1261/rna.066092.118. Epub 2018 Apr 12.

Abstract

N6-threonyl-carbamoyl adenosine (t6A) is a universal tRNA modification found at position 37, next to the anticodon, in almost all tRNAs decoding ANN codons (where N = A, U, G, or C). t6A stabilizes the codon-anticodon interaction and hence promotes translation fidelity. The first step of the biosynthesis of t6A, the production of threonyl-carbamoyl adenylate (TC-AMP), is catalyzed by the Sua5/TsaC family of enzymes. While TsaC is a single domain protein, Sua5 enzymes are composed of the TsaC-like domain, a linker and an extra domain called SUA5 of unknown function. In the present study, we report structure-function analysis of Pyrococcus abyssi Sua5 (Pa-Sua5). Crystallographic data revealed binding sites for bicarbonate substrate and pyrophosphate product. The linker of Pa-Sua5 forms a loop structure that folds into the active site gorge and closes it. Using structure-guided mutational analysis, we established that the conserved sequence motifs in the linker and the domain-domain interface are essential for the function of Pa-Sua5. We propose that the linker participates actively in the biosynthesis of TC-AMP by binding to ATP/PPi and by stabilizing the N-carboxy-l-threonine intermediate. Hence, TsaC orthologs which lack such a linker and SUA5 domain use a different mechanism for TC-AMP synthesis.

Keywords: Sua5; TsaC; t6A37; tRNA modification; threonylcarbamoyl adenosine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / analogs & derivatives*
  • Adenosine / biosynthesis
  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / metabolism
  • Adenosine Triphosphate / metabolism
  • Amino Acid Motifs
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism
  • Models, Molecular
  • Mutation
  • Protein Conformation
  • Protein Domains
  • Pyrococcus abyssi / enzymology*
  • RNA, Transfer / chemistry
  • RNA, Transfer / metabolism*
  • Structure-Activity Relationship

Substances

  • Archaeal Proteins
  • N(6)-(N-threonylcarbonyl)adenosine
  • Adenosine Triphosphate
  • RNA, Transfer
  • Adenosine Triphosphatases
  • Adenosine