Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
Structural analyses by NMR reveal insights into the enzymatic mechanism of tRNA t6A modification biosynthesis
Posttranscriptional modification is an inherent feature of RNA maturation and is particularly abundant in transfer RNA (tRNA), where over 90 distinct modifications are found across all phylogenetic groups. Of these, the universally conserved N6-threonylcarbamoyladenosine (t6A) modification 3’-adjacent to the anticodon is essential in translational fidelity for several tRNA species by ensuring accurate codon recognition.
Recent studies of the t6A biosynthesis pathway in bacteria have identified four proteins that are necessary and sufficient for in vitro formation: YrdC, YgJD, YjeE and YeaZ. As this enzyme complex is comprised of essential proteins, two of which are unique to bacteria, YeaZ and YjeE, it is a potential target for antimicrobial development. Thus, a detailed understanding of the interactions within this pathway holds significant value but has yet to be accomplished; therefore, further structural characterization and biochemical analyses are necessary.
Of particular interest is YrdC, a universally conserved, monomeric 21 kDa protein that is believed to be the central component of this complex. Using quantitative methods, we have shown that YrdC binds to all components required for t6A biosynthesis: ATP, L-threonine and the unmodified target tRNA. Additionally, we have developed a novel application of saturation transfer difference (STD-) NMR for detecting protein-RNA interactions and used it to observe the interaction of YrdC with a 17-nt tRNALys anticodon domain truncation.
In order to map these binding interfaces under biological conditions, high-resolution structures are vital; therefore, the solution structure of E. coli YrdC was determined by NMR. Several key residues have been identified by 15N-HSQC titrations and molecular docking simulations were performed to map ligand binding sites and protein-protein interaction interfaces.
This work is supported by NSF grant MCB1101859.
Kimberly A. Harris1,2
1North Carolina State University