Figure 5: Tsr1 excludes binding of eIF5B and Rio1.

(a) The Tsr1 structure was fitted into aligned, previously published cryo-EM maps of pre-40S particles derived from wild-type (transparent magenta, EMD1926) and Tsr1-depleted (opaque white, EMD1927) yeast cells. The particle is viewed from the mRNA-binding surface (left) and a 90° rotation (right) to show the positioning of Tsr1 with respect to the 60S interaction surface. Tsr1 is shown with the front view rotated ∼180° in the plane of the page with respect to Fig. 1c. Inset figures indicate the electrostatic surface of Tsr1 when fitted into the particle. (b) A 40S structure from a mammalian pre-initiation complex, including eIF5B (PDBID 4UJD), was fitted to EMD1926. Proteins, rRNA and tRNA from the pre-initiation complex are hidden, with the exception of eIF5B, for the sake of clarity. The orientations are as in a. The face of the 40S that interacts with the 60S is indicated with a bracket. (c) Side view of Tsr1 fitted to cryo-EM particles with 40S subunit from yeast fitted into the map (PDBID 4V8Y). Bases previously identified as Rio1 crosslinking sites by CRAC are coloured green and shown as space-fill. (d) Overview of the arrangement of Rio1 (green), Tsr1 (purple) and common (black) CRAC sites. (e) Tsr1 binding is mutually exclusive with binding of eIF5b and Rio1, and with 60S subunit joining. We speculate that negative charges on the surface of Tsr1 prevent premature 60S joining. Displacement of Tsr1, along with other early and intermediate pre-40S assembly factors (Rio2, Ltv1, Enp1 and Dim1), is required for late pre-40S maturation. Following removal of Tsr1, eIF5B-binding promotes GTP-dependent 60S subunit association, while Rio1 is predicted to promote ATP-dependent association. In both cases, formation of these 80S-like particles may constitute a quality control step, verifying that the pre-40S particle is close to the mature structure, before final 3′ processing of the 18S rRNA by the endonuclease Nob1.