The ribosome-associated complex RAC serves in a relay that directs nascent chains to Ssb

The conserved ribosome-associated complex (RAC) consisting of Zuo1 (Hsp40) and Ssz1 (non-canonical Hsp70) acts together with the ribosome-bound Hsp70 chaperone Ssb in de novo protein folding at the ribosomal tunnel exit. Current models suggest that the function of Ssz1 is confined to the support of Zuo1, however, it is not known whether RAC by itself serves as a chaperone for nascent chains. Here we show that, via its rudimentary substrate binding domain (SBD), Ssz1 directly binds to emerging nascent chains prior to Ssb. Structural and biochemical analyses identify a conserved LP-motif at the Zuo1 N-terminus forming a polyproline-II helix, which binds to the Ssz1-SBD as a pseudo-substrate. The LP-motif competes with nascent chain binding to the Ssz1-SBD and modulates nascent chain transfer. The combined data indicate that Ssz1 is an active chaperone optimized for transient, low-affinity substrate binding, which ensures the flux of nascent chains through RAC/Ssb.


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For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Irmgard Sinning, Sabine Rospert February 25, 2020 Diffraction data were measured under cryogenic conditions (100 K; Oxford Cryosystems Cryostream) at the European Synchrotron Radiation Facility (ESRF, Grenoble) beamline id30a3.
Data were processed with XDS. Phases were obtained by molecular replacement using PHASER in the CCP4I2 software package. Iterative model building and refinement were done with COOT and phenix.refine . The validation was performed using EDSTATS and MOLPROBITY included in CCP4I2. UCSF chimera was used for figure preparation. Sequence alignments were performed using Clustal Omega and visualized with ESPript 3.0. In addition, the following software was used in this study: Python version 3.6; Graphpad Prism version 6.07; AIDA ImageAnalyzer version 4.27.
Accession codes: Coordinates and structure factors have been deposited in the Protein Data Bank under accession code 6SR6. The PDB datasets 5MB9, 1DKZ and 4EZP have been used in this study. The source data underlying Figures 1c-f, 2b-e, 3b-f, and Supplementary Figures 1a-j, 5a-c, and 6a,b are provided as a Source Data file. Other data that support the findings of this study are available from the corresponding authors on reasonable request.