YrdC exhibits properties expected of a subunit for a tRNA threonylcarbamoyl transferase

Abstract

The post-transcriptional nucleoside modifications of tRNA's anticodon domain form the loop structure and dynamics required for effective and accurate recognition of synonymous codons. The N 6 -threonylcarbamoyladenosine modification at position 37 (t 6 A 37 ), 39-adjacent to the anticodon, of many tRNA species in all organisms ensures the accurate recognition of ANN codons by increasing codon affinity, enhancing ribosome binding, and maintaining the reading frame. However, biosynthesis of this complex modification is only partially understood. The synthesis requires ATP, free threonine, a single carbon source for the carbamoyl, and an enzyme yet to be identified. Recently, the universal protein family Sua5/YciO/YrdC was associated with t . YrdC also demonstrated specificity toward the unmodified versus modified anticodon pentamer UUUUA and toward threonine and ATP. The protein did not significantly alter the ASL architecture, nor was it able to base flip A 37 , as determined by NMR, circular dichroism, and fluorescence of 2-aminopuine at position 37. Thus, current data support the hypothesis that YrdC, with many of the properties of a putative threonylcarbamoyl transferase, most likely functions as a component of a heteromultimeric protein complex for t 6 A 37 biosynthesis.

NMR-based Structural Analysis of Threonylcarbamoyl-AMP Synthase and Its Substrate Interactions

“…TsaC has been shown to interact with three small substrates, L-threonine (25), ATP (24), and CO 2 /HCO 3 Ϫ (17), as well as hypomodified tRNA (24,25,27), TsaD and TsaB (16). The synthesis of TC-AMP, the central role of TsaC in t 6 A 37 biosynthesis, requires a number of intermolecular interactions and enzymatic activity.…”

“…L-Threonine-binding Site-E. coli TsaC and S. tokodaii Sua5 have been shown to exhibit specificity for L-threonine over other amino acids (25,50), and E. coli TsaC has been shown to catalyze TC-AMP formation (17). Therefore, we sought to characterize the TsaC/L-threonine binding interface to provide insight into structural aspects of the enzymatic mechanism.…”

“…TsaC Interaction with ATP-E. coli TsaC has been shown to bind ATP and L-threonine to produce TC-AMP (16,24,25). The interaction of TsaC with ATP was characterized by isothermal titration calorimetry (ITC; Fig.…”

“…TsaC is essential in E. coli, but Sua5 is not essential in S. cerevisiae (24), even though both appear to have similar roles in t 6 A 37 biosynthesis. E. coli TsaC selectively binds both ATP and L-threonine (24,25) and is the sole protein of the t 6 A-synthase complex capable of L-threonine-dependent conversion of ATP to AMP (16). More recently, both TsaC and its B. subtilis homolog, YwlC, have been shown to catalyze the formation of the activated precursor L-threonylcarbamoyl-AMP (TC-AMP) from L-threonine, ATP, and CO 2 /HCO 3 Ϫ (Fig.…”

“…The argument is strengthened by the ability of P. abyssi Kae1, the TsaD homolog, to bind TC-AMP and catalyze the transfer to tRNA (26). However, several studies have shown that E. coli TsaC selectively binds hypomodified tRNA (24,25,27), indicating TsaC may have a more substantial, diverse role in bacteria than in eukaryotes because S. cerevisiae Sua5 does not exhibit RNA binding activity (18).…”