Poster abstracts

Poster number 14 submitted by Brianne Sanford

Mapping the interaction between bacterial YbaK and tRNAPro, a novel trans editing complex

Brianne Sanford (Department of Chemistry and Biochemistry, Center for RNA Biology), Eric Danhart (Ohio State Biochemistry Program), Maryanne Refaei (Ohio State Biochemistry Program), Mark Foster (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State Biochemistry Program), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State Biochemistry Program)

Abstract:
Prolyl-tRNA synthetase (ProRS) is a class II synthetase that aminoacylates proline onto cognate tRNA^Pro. ProRSs commonly misactivate alanine and cysteine, which must be edited to avoid incorporation into the translating protein. The so-called “triple-sieve” editing mechanism used by ProRS involves a cis editing domain on ProRS that hydrolyzes Ala-tRNA^Pro and the single-domain YbaK protein, which edits mischarged Cys-tRNA^Pro in trans. Previous fluorescence anisotropy studies suggested that YbaK and ProRS interact in vitro and the binding affinity is increased in the presence of tRNA^Pro. Additionally, chemical cross-linking studies support the formation of a ProRS/YbaK/tRNA^Pro ternary complex in vitro. Recent work examining these interactions in vivo using a split-GFP reassembly assay (B.R. So, PhD Thesis, 2010) and an independent cross-linking approach (L. Nie and T. Magliery, unpublished), support YbaK/ProRS complex formation. Although these studies indicate that YbaK, ProRS, and tRNA^Pro interact as a ternary complex, there is currently no high-resolution, three-dimensional structure of this interaction. The present work is focused on defining the interaction between tRNA^Pro and YbaK at the molecular level. Two dimensional ¹⁵N-H NMR spectra of ¹⁵N-enriched YbaK have been obtained in the absence and presence of tRNA^Pro. Preliminary data indicate that a subset of peaks shift or change in intensity upon tRNA^Pro binding and assignment of these peaks is currently underway. In addition to the NMR studies, footprinting and cross-linking studies are being conducted to define the tRNA^Pro/YbaK interaction interface. These studies in combination with computational docking will yield the first three-dimensional model of this trans editing complex.

References:
An, S. and K. Musier-Forsyth, Cys-tRNA^Pro editing by Haemophilus influenzae YbaK via a novel synthetase/YbaK/tRNA ternary complex. J Biol Chem, 2005. 280(41): p. 34465-72

Keywords: tRNA, aminoacyl tRNA synthetase