Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
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 N6-threonylcarbamoyladenosine modification at position 37 (t6A37), 3’-adjacent to the anticodon, of many tRNA species ensures the accurate recognition of several ANN codons by increasing codon affinity and enhancing ribosome binding1,2. Considerable data exists on the biophysical aspects of t6A37, however, the biosynthesis pathway of this hypermodified base is only partially understood3. This pathway requires ATP, threonine, a carbon source, and possibly the universal protein family YrdC/Sua5, which has been shown to be involved in t6A37 biosynthesis4,5. To further investigate this possibility, we examined the interaction of E. coli YrdC with the heptadecamer anticodon stem loop of tRNA lysine (ASLLys). As determined by mass spectrometry analysis, NMR, and quenching of intrinsic fluorescence, YrdC bound unmodified ASLLys with high affinity (Kd = 0.27 ± 0.20 µM), t6A37-modified ASLLys with significantly lower affinity (Kd = 1.36 ± 0.39 µM), and showed specificity toward threonine and ATP. YrdC also preferentially binds threonine over other amino acids tested by STD-NMR. Our studies of the YrdC-ASLLys interaction by NMR, CD, and fluorescence of 2-aminopuine at position 37 of ASLLys, indicated no structural change in the RNA. Therefore, catalytic function appears to be limited under these in vitro conditions and YrdC is most likely a subunit of a t6A37 synthetase complex. Further characterization of the protein-protein and protein-RNA interactions will provide information to determine the role of YrdC in t6A37 biosynthesis.
Kimberly A. Harris1,2
1Department of Molecular & Structural Biochemistry