Fig. 1: Zuo1 and Ssz1 contact nascent chains prior to Ssb.
From: The ribosome-associated complex RAC serves in a relay that directs nascent chains to Ssb
a Domain structure of yeast Ssb, Zuo1, and Ssz1. Nucleotide binding domain (NBD), linker, substrate binding domain β (SBDβ), substrate binding domain α (SBDα), N-terminal domain (N), J-domain (J), Zuo1 homology domain (ZHD), middle domain (MD), and four-helix bundle (HD). b Experimental set up of crosslinking experiments. Isolated RNCs carrying [35S]-labelled nascent chains are crosslinked to adjacent proteins using the homobifunctional amino-reactive crosslinker BS3, spacer length 11.4 Å. Crosslink products between nascent chains and Ssb, Ssz1, or Zuo1 are then identified via immunoprecipitation under denaturing conditions using antibodies directed against Ssb, Ssz1, or Zuo1. For details see Methods. c Contacts of Ssb, Zuo1, and Ssz1 with short nascent chains. RNCs carrying Pgk1-40 (40 residues), -45 (45 residues), or -50 (50 residues) were generated in a wild type translation extract as described in (b). Crosslink products (Pgk1-XL) between Ssb and nascent Pgk1 (Ssb); Zuo1 and nascent Pgk1 (Zuo1); and Ssz1 and nascent Pgk1 (Ssz1). d Zuo1 and Ssz1 interact with nascent Pgk1-100 (100 residues) in the absence of ribosome-bound Ssb. The experiment was performed as described in c using RNCs from wild type, Δssb1Δssb2 (Δssb), Δssb1Δssb2Δzuo1Δssz1 (ΔssbΔrac), or Ssb1-ΔC23 translation extract. The green asterisk indicates the crosslink between Ssb and Pgk1-100 in the total (d–f). e Transfer of the nascent chain from Zuo1 to Ssb requires Ssz1. The experiment was performed as in (c) using RNCs from Δzuo1Δssz1Δssb1Δssb2 translation extract. Ribosome-free extracts (upper panel) obtained from wild type (Ssb, Zuo1, Ssz1), Δzuo1 expressing Zuo1-ΔN49 (Ssb, Zuo1-ΔN49, Ssz1), Δssz1 (Ssb, Zuo1), Δzuo1Δssz1 (Ssb) were added after completion of translation reactions, prior to crosslinking. Rebinding of Ssb to RNCs was analyzed as described in Supplementary Fig. 1f. f Ssb prevents crosslinking of Zuo1 and Ssz1 to long nascent chains. The experiment was performed as in (c) using RNCs from wild type (wt) or Δssb1Δssb2 (Δssb) translation extract. Purified Ssb1 (Supplementary Fig. 1g,h) was added prior to the translation reaction as indicated (+ Ssb). Source data for (c–e) are provided as a Source Data file.
