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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