NMR and MS Studies on Eukaryotic Release Factor 1 — ASN Events
  1. Schedule
  2. Session Seven - Poster Session B Including Happy Hour & Trade Display
  3. NMR and MS Studies on Eukaryotic Release Factor 1

NMR and MS Studies on Eukaryotic Release Factor 1 (#234)

Suzana Markolovic 1 , Ivanhoe K. H. Leung 1 2 , Timothy D. W. Claridge 1 , Sarah E. Wilkins 1 , Christopher J. Schofield 1
  1. Department of Chemistry, University of Oxford, Oxford, United Kingdom
  2. School of Chemical Sciences, The University of Auckland, Auckland, New Zealand

In eukaryotes, translation termination is mediated by a release factor complex that comprises of eukaryotic release factors 1 and 3 (ERF1/3), which act cooperatively to ensure efficient stop codon recognition and effective peptidyl-tRNA ester bond hydrolysis.1 ERF1 is a multi-domain protein, with the N-domain (ERF1-N) being involved in the recognition of all three stop codons.2 Previous studies have revealed that ERF1-N possesses a conserved NIKS motif;3 hydroxylation of the lysine residue (K63) within this motif is required for optimal translation termination efficiency.4 Lysyl hydroxylation of ERF1 is catalysed by Jumonji domain-containing protein 4 (JMJD4), which belongs to a superfamily of Fe(II)-dependent oxygenase that utilise 2-oxoglutarate (2OG) as a cosubstrate.4 However, the exact molecular mechanism by which hydroxylation of ERF1-N influences translation termination is poorly understood. We postulate that a post-translational modification like hydroxylation may induce changes in the structure and dynamics of ERF1-N, which in turn may regulate intermolecular interactions (e.g. protein/protein or protein/RNA interactions) and hence translation termination efficiency.

Here, using mass spectrometry (MS), we discovered a second JMJD4-catalysed post-translational modification of ERF1-N. Using protein NMR spectroscopy, the effects of these post-translational modifications on the structure and dynamics of ERF1-N were also studied. Finally, the interactions between unmodified and modified ERF1-N and ribosomal RNA were also investigated.

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  1. Kisselev, L. L.; Buckingham, R. H. Translational termination comes of age. Trends Biochem. Sci. 2000, 25, 561–566.
  2. Song, H.; Mugnier, P.; Das, A. K.; Webb, H. M.; Evans, D. R.; Tuite, M. F.; Hemmings, B. A.; Barford, D. The crystal structure of human eukaryotic release factor eRF1-mechanism of stop codon recognition and peptidyl-tRNA hydrolysis. Cell 2000, 100, 311–321.
  3. Frolova, L.; Seit-Nebi, A.; Kisselev, L. E. V. Highly conserved NIKS tetrapeptide is functionally essential in eukaryotic translation termination factor eRF1. RNA 2002, 8, 129–136.
  4. Feng, T.; Yamamoto, A.; Wilkins, S. E.; Sokolova, E.; Yates, L. A.; Munzel, M.; Singh, P.; Hopkinson, R. J.; Fischer, R.; Cockman, M. E.; Shelley, J.; Trudgian, D. C.; Schodel, J.; McCullagh, J. S. O.; Ge, W.; Kessler, B. M.; Gilbert, R. J.; Frolova, L. Y.; Alkalaeva, E.; Ratcliffe, P. J.; Schofield, C. J.; Coleman, M. L. Optimal translational termination requires C4 lysyl hydroxylation of eRF1. Mol. Cell 2014, 53, 645–654.