Disentangling conformational and compositional heterogeneity

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ABSTRACT It has recently been discovered that “specialized” eukaryotic ribosomes enriched or depleted in certain core ribosomal proteins (RPs) preferentially translate groups of mRNA sequences, and moreover that these groups of mRNAs often relate to specific cellular pathways ​(Shi et al., 2017)​. This link between ribosome compositional heterogeneity and genome regulation has been studied by mass spectrometric and sequencing analyses of ribosomes and mRNA sequences isolated from distinct ribosome populations. However, simple rules linking “specialized” ribosomes and sequence preferences have not yet emerged. Work by our collaborator Maria Barna’s lab showed that the presence or absence of RPS25/eS25, a substoichiometric RP located near the mRNA exit tunnel on the 40S subunit of the eukaryotic ribosome, affects translation of ~150 genes. Our hypothesis is that this core RP imparts recognition of mRNAs by altering the structure and conformational dynamics of the ribosome. To address this question, we will isolate and characterize ribosomes with or without RPS25/eS25 by cryoEM. Due to its fundamentally single particle nature, cryoEM is an optimal method for examining large heterogeneous macromolecular machines. However, at present the end result of cryoEM analysis is a single structure reflecting the average of all contributing conformations. We propose to develop a new class of model that incorporates the concept of a dynamic structure with conformational heterogeneity. We will collaborate with the developers of cisTEM to parameterize flexibility of a structural model and to iteratively refine model motions alongside the canonical structure during refinement of cryoEM maps. We will refine flexible models of ribosomes with and without RPS25/eS25 using this new functionality to discover a link between structure and function, revealing the atomic mechanisms of translation specificity and implicating a general mechanism for genome regulation by ribosome composition. In summary, we will develop new structure refinement capabilities for cryoEM that encode structural flexibility and apply them to the discovery of differences in flexibility between ribosomes preferential to different mRNA sequences. This study will be a proof of principle for the discovery of biologically relevant structural motions by single particle cryoEM, and the expanded ribosome and mRNA models will suggest a mechanism for genome regulation at the stage of protein synthesis.