Significance of Knotted Structures for Function of Proteins and Nucleic Acids - September 17-21, 2014

Significance of Knotted Structures for Function of Proteins and Nucleic Acids

Poster Session II

51 – POS Board 23 The Structural Basis of tRNA t 6 A Modification in Bacteria Wenhua Zhang , Bruno Collinet, Herman Van Tilbeurgh. University Paris-Sud 11, Orsay, France.

The universal and essential N 6 -threonylcarbamoyladenosine (t 6 A) modification occurring on ANN-decoding tRNAs plays a pivotal role in translational fidelity. The enzymes responsible for t 6 A modification of tRNA had remained a mystery for 40 years until recently, it was discovered that the Kae1/Qir7/YgjD and Sua5/YrdC protein families are related and responsible for biosynthesis of t 6 A at cellular level. In archaea and eukarya, t 6 A is biosynthesized by the KEOPS/EKC protein complex (Kae1, Bud32, Cgi121 and Pcc1 in archaea, with a fifth Gon7 specific in yeast) and in mitochondria by Qri7 (a KAE1 paralog) in conjunction with Sua5. In bacteria, the biosynthesis of t 6 A is accomplished by YgjD, YeaZ, YjeE and YrdC using tRNA, ATP, L -threonine and bicarbonate as substrates. We used molecular cloning, biochemical assays, X-ray crystallography and SAXS to elucidate the molecular mechanism underscoring the tRNA t 6 A modification in bacteria. We show that the YgjD-YeaZ-YjeE ternary complex forms in presence of ATP and is dynamically tuned by both the heterodimer YgjD-YeaZ and the hydrolysis of ATP into ADP by ATPase YjeE that is characteristic of GTPase proteins. Our crystal structure of dimer YgjD- YeaZ shows that dimerization of YgjD and YeaZ induces a conformational change on YeaZ in proximity to the interface and thereby creates an atypical ADP-binding site. The activation of the ATPase activity of YjeE by heterodimer YgjD-YeaZ is reminiscent of GTPase Activating Proteins (GAPs). Guided by the similar orientation of ADPs bound in crystal structures of YjeE and in heterodimer YgjD-YeaZ, we built a model for the YgjD-YeaZ-YjeE ternary complex as validated by SAXS. The tRNA t 6 A modification assay in vitro shows that the YgjD-YeaZ-YjeE ternary complex is essential for the biosynthesis of t6A in conjunction with YrdC while the ATPase activity of YjeE is dispensable.

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