Proteins must assemble into their proper three dimensional shape to correctly function. Misfolding and aggregation are associated with a variety of devastating human diseases, such as neurodegeneration and cancers. Folding can begin as soon as a nascent protein extends out of the exit tunnel of the ribosome and continues after synthesis is complete. Chaperones are a functional class of proteins that aid in the folding and assembly of client proteins. One functional network, consisting of multiple chaperone families, is linked to protein synthesis, with some components acting upon the translating nascent chain. The ribosome-associated complex (RAC) binds near the ribosome exit tunnel, and it recruits and regulates members of the 70 kDa heat-shock protein (Hsp70) family of chaperones. The nascent-chain associated complex (NAC), another CLIP, also binds near the exit tunnel. The role of NAC is less clear, but it does appear to be involved in RAC and Hsp70 activity. Here, the roles of yeast NAC and RAC in cotranslational protein folding will be investigated for every cellular protein. Yeast has multiple genes that encode the dimeric NAC, and the various combinations of these gene products associate with different sets of mRNA transcripts during translation. A selective ribosome profiling strategy will determine the nature o the interaction between NAC and the translating ribosome with nucleotide-level resolution. Sequence elements that correlate with particular NAC isoforms will be discovered, and the kinetics of NAC recruitment over the course of translation will be revealed. Selective profiling of the ribosome-recruited Hsp70 in yeast, SSB, will indicate the initial points of association of this chaperone to the nascent chain. A peptide tiling array will confirm these interactions. In a second aim, the role of NAC in the RAC-mediated recruitment of SSB will determined. A microarray assay will identify mRNA transcripts that associate with SSB in NAC deletion strains. Comparing to wild-type association will reveal which transcripts rely upon NAC for proper Hsp70 recognition. The assay will also be used to describe the partial rescue of SSB deletion by the major cytosolic Hsp70 homologs, SSA, and whether this rescue is also NAC-mediated. Protein-protein interaction assays will decipher the interactions between the nascent chain, NAC, RAC, and SSB. These interaction assays will provide a platform to test mechanistic hypotheses. Together, this work will provide a unified model for nascent chain recognition and engagement by the ribosome-associated cotranslational chaperone network.
Neurodegenerative disease, cancer, and aging result when newly made proteins do not properly assemble. The research proposed in this training program will elucidate the initial engagement of dedicated protein folding pathways while a new protein is being synthesized. Through an understanding of the mechanism of early folding events, we can better explain, predict, and ultimately treat misfolding diseases.
|Pechmann, Sebastian; Chartron, Justin W; Frydman, Judith (2014) Local slowdown of translation by nonoptimal codons promotes nascent-chain recognition by SRP in vivo. Nat Struct Mol Biol 21:1100-5|