Structural characteristics of E. coli YrdC suggest a role in the enzymatic biosynthesis of the tRNA modification N6‐threonylcarbamoyladenosine

Nucleoside modifications are vital for the proper structure and function of tRNA. The N 6 ‐threonylcarbamoyladenosine modification at position 37 (t 6 A 37 ), 3′‐adjacent to the anticodon, of many tRNA species ensures the accurate recognition of several ANN codons by increasing codon affinity and enhancing ribosome binding. Considerable data exists on the biophysical aspects of t 6 A 37 . However, the biosynthesis pathway of this hypermodified base is only partially understood. This pathway requires ATP, threonine, a carbon source, and possibly the universal protein family YrdC/Sua5, which has been shown to be involved in t 6 A 37 biosynthesis. To further investigate this possibility, we examined the interaction of E. coli YrdC with the heptadecamer anticodon stem loop of tRNA lysine (ASL Lys ). As determined by quenching of intrinsic fluorescence, YrdC bound unmodified ASL Lys with high affinity (K d =0.27±0.20μM), t 6 A 37 ‐modified ASL Lys with significantly lower affinity (K d =1.36±0.39μM), and showed specificity toward threonine and ATP. Our studies of the YrdC‐ASL Lys interaction by NMR, CD, and fluorescence of 2‐aminopuine at position 37 of ASL Lys , indicated no structural change in the RNA. Therefore, its function appears to be limited under these in vitro conditions. To address this, the structural interaction of YrdC with t 6 A 37 biosynthesis components under reaction conditon is under investigation. We have solved the solution structure by NMR of YrdC (21 kDa) and ASL Lys , separately. NMR analysis of YrdC in complex with ASL Lys , threonine, and ATP is underway. Structural characterization of this protein‐RNA complex will provide information to determine the enzymatic mechanism of YrdC.