Tidsupplöst kryoelektronmikroskopi för studier av den genetiska kodens översättning vid cellfri proteinsyntes med jäst- och bakteriekomponenter
Tidsperiod: 2017-07-01 till 2020-06-30
Projektledare: Jingji Zhang
Bidragstyp: Bidrag för anställning eller stipendier
Budget: 3 150 000 SEK
I plan to use the new technique “time-resolved cryo-electron microscopy (TR cryo-EM)” for structural and kinetics analyses of accuracy of genetic code translation into proteins by transfer RNAs (tRNAs) and protein factors. TR cryo-EM allows for near-atomic resolution of ribosomal complexes in transient states of code reading combined with kinetics analysis based on the time dependent appearance and disappearance of multitudes of singular ribosome complexes rapidly vitrified on the cryo-EM grid at different times in the peptide elongation cycle.I will apply TR cryo-EM and single-particle reconstruction to obtain high-resolution structures of ribosomes in tRNA- and release factor-bound complexes of bacteria (18 months) and yeast (18 months), to assess and compare the accuracy of genetic code translation in prokaryotes and eukaryotes. I will, in particular, address the question if the ribosome uses two energy driven proofreading steps to amplify the accuracy of sense codon reading in bacteria and yeast and the role of protein factor eRF3 for accurate termination in yeast. The experiments will be performed in Prof. J. Frank’s laboratory at Columbia University (NY), where TR cryo-EM has been perfected.In the proposed project I will complement my previous training in biochemistry, thermodynamics and fast kinetics as applied to the bacterial ribosome with structural biology where cryo-EM is fully integrated with kinetics based on ensembles of singular ribosome complexes.