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 N⁶-threonylcarbamoyladenosine modification at position 37 (t⁶A₃₇), 3′-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 t⁶A₃₇ biosynthesis. To further investigate the role of YrdC in t⁶A₃₇ biosynthesis, the interaction of the Escherichia coli YrdC with a heptadecamer anticodon stem and loop of lysine tRNA (ASL^Lys_UUU) was examined. YrdC bound the unmodified ASL^Lys_UUU with high affinity compared with the t⁶A₃₇-modified ASL^Lys_UUU (K_d = 0.27 ± 0.20 μM and 1.36 ± 0.39 μM, respectively). 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 A₃₇, 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 t⁶A₃₇ biosynthesis.