Visualizing a dynamically tethered DEAD-box ATPase-rRNA complex during ribosome maturation-
EMSL Project ID
51013
Abstract
The assembly of ribosomes is a critical process in cells, and its dysregulation contributes to a range of human diseases termed ribosomopathies. Ribosome maturation relies on numerous enzymes, most prominently on a number of ATPases of the DEAD-box family. We are using an integrated, structure-driven effort to define the mechanistic details of eukaryotic ribosome biogenesis, with a specific focus on the large (or 60S) subunit. Eukaryotes possess more than 200 ribosomal biogenesis factors (BFs) that drive the unidirectional maturation of ribosomes. We have generated trapping mutants of specific DEAD-box proteins, allowing us to isolate and structurally characterize two distinct maturation intermediates of the large or 60S ribosomal subunit. Mass spectrometry analysis shows that the DEAD-box ATPase Drs1 is abundantly present in only one of these isolated complexes, suggesting an important role for this protein in the rRNA rearrangements observed between these states. Because no obvious Drs1 density was observed in our reconstructions, we used cross-linking mass-spectrometry to define Drs1 interactions partners and used recombinant factors to characterize a specific interaction between the C-terminal domain of Drs1 and the C-terminus of the Nsa1 biogenesis factor in vitro. These findings suggest that Drs1 is part of a loosely tethered rRNP element which includes the Erb1 and Ytm1 biogenesis factors, and rRNA domain III. This assembly cannot engage the 60S core until Drs1 is removed, suggesting that Drs1 prevents the premature docking of this subdomain during maturation. In order to visualize this dynamic element, we have deleted disordered regions in the Nsa1 tether to reduce the conformational flexibility of the Drs1 subcomplex. Preliminary reconstructions validate this approach, revealing new features in our cryo-EM maps. Because of the dynamic nature of this element, we propose to collect a large dataset to reveal structural features of this pre-docking structure that will reveal, for the first time, details of elusive dynamic elements in 60S biogenesis intermediates.
Project Details
Start Date
2019-09-24
End Date
2020-06-24
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members