ABSTRACT
Bacterial and eukaryotic tRNAs that decode codons starting with uridine have a hydrophobically hypermodified adenosine at position 37 (A37) adjacent to the 3'-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. However, it remains unclear as to how the corresponding tRNAs are selected to be modified by alkylation at the correct position of the adenosine base. We have determined a series of crystal structures of bacterial tRNA isopentenyltransferase (MiaA) in apo- and tRNA-bound forms, which completely render snapshots of substrate selections during the modification of RNA. A compact evolutionary inserted domain (herein swinging domain) in MiaA that exhibits as a highly mobile entity moves around the catalytic domain as likely to reach and trap the tRNA substrate. Thereby, MiaA clamps the anticodon stem loop of the tRNA substrate between the catalytic and swinging domains, where the two conserved elongated residues from the swinging domain pinch the two flanking A36 and A38 together to squeeze out A37 into the reaction tunnel. The site-specific isopentenylation of RNA is thus ensured by a characteristic pinch-and-flip mechanism and by a reaction tunnel to confine the substrate selection. Furthermore, combining information from soaking experiments with structural comparisons, we propose a mechanism for the ordered substrate binding of MiaA.
FIGURE CAPTION
Overall structure of the E. coli MiaA-tRNA complex at 2.55A. Front-view ribbon representation of the complex in stereo view, depicted in different colors for each domain: yellow and red for the catalytic domain and the swinging domain, respectively. The bound tRNAPhe (GAA) is shown in sky blue for its nucleosides and in magenta for its phosphate backbones. The flipped-out target A37 is emphasized by the dotted spheres. The Mg2+ ion is drawn as green spheres. These color schemes are used throughout, unless otherwise noted.
REFERENCES
Snapshots of Dynamics in Synthesizing N6-Isopentenyladenosine at the tRNA Anticodon.
Sarin Chimnaronk, Farhad Forouhar, Junichi Sakai, Min Yao, Cecile M. Tron, Mohamed Atta, Marc Fontecave, John F. Hunt, and Isao Tanaka.
Biochemistry 48, 5057-5065 (2009)