Coordination of mRNA translation with the action of ribosome-associated chaperones in yeast

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Folding of newly synthesized proteins is of fundamental importance for the functioning of all cells. It is supported by a network of molecular chaperones, some of which act co-translationally by engaging the translating ribosome and the nascent chain. To efficiently support the folding process and coordinate chaperone activity with the action of other protein maturation factors, nascent chain engagement with nascent chains must be precisely synchronized with the progression of translation. The proposed project aims to unravel mechanistic principles of the coordination of the protein translation and folding machineries, by studying the function of the ribosome associated chaperone triad in S. cerevisiae. This triad consists of the Hsp70 chaperone Ssb1,2 (Ssb) and the heteromeric RAC complex composed of the Hsp40 chaperone Zuo1 and the Hsp70 chaperone Ssz1.The first major aim of the proposal is to analyze the coordinative role of RAC on Ssb-mediated chaperoning of nascent polypeptides. Using Selective Ribosome Profiling (SeRP) as major experimental approach, we will identify the nascent chain interactome of RAC at near-codon resolution in vivo. By performing bioinformatic analysis of RAC interaction profiles we will identify nascent chain features that mediate RAC binding. SeRP in wild type and chaperone mutant strains, supported by in vitro binding studies of purified chaperones with translating ribosomes, will help exploring the functional interplay of RAC and Ssb and reveal how nascent chain binding of RAC contributes to the action of Ssb in vivo. The second major aim of the proposal investigates the coordination of RAC function with translation. We will test whether and how RAC binding impacts the kinetics of translation and also test the alternative model, that variation of translation elongation guides chaperone function. We will perform run-off ribosome profiling to determine mRNA-specific translation speed of ribosomes in wild type and chaperone mutant strain backgrounds and explore the prevalence of colliding ribosomes (disomes) on mRNAs as an indicator of local ribosome pausing. Correlating chaperone binding profiles with the data exploring translation kinetics will reveal whether translation speed changes correlate with Ssb and RAC action at the ribosome. To assess the mechanism coordinating translation speed and chaperone function, we will test a possible direct impact of RAC binding on tRNA binding and translation elongation and investigate whether ribosome pausing, conferred by features within mRNAs or nascent chains, may facilitate the association of chaperones with translating ribosomes.Together, these analyses will provide new insights into the intricate mechanism coordinating Hsp70- assisted co-translational protein folding with translation and improve our conceptual understanding how protein maturation is coupled to synthesis.