Assess how variations in ribosome composition structurally regulate translation.
Central to dissecting the common mechanisms of translational regulation by specialised ribosomes, and something currently missing for most specialised ribosomes, is an understanding of the structural impact of specialisation on translation regulation. This will be addressed in Theme 3, which, when combined with Themes 2 and 3, will allow us to unravel the molecular mechanisms of ribosome specialisation.
Our established structural approach to characterising specialised ribosomes involves: 1) purifying candidate ribosomes and determining their protein composition, including the identity and stoichiometry of protein subunits that confer specialisation, by quantitative MS (e.g. TMT-MS), and 2) characterising their structure by cryo-EM, to provide insight into the structural differences that drive specialisation. This workflow has allowed us to unravel composition and structural differences in ribosomal paralogs between Drosophila ribosomes isolated from testes and ovaries, and we will use it during the sLoLa to gain structural information on the specialised ribosomes we will purify. To obtain further information on regions of the ribosome that have been historically challenging, we will adapt structural MS approaches for the study of ribosomes, including native MS, which allows unravelling the stoichiometry and composition of specialised ribosomes, and cross-linking MS, which allows probing for structural/dynamical changes in the ribosome.
