Fig. 10.   Models for individual CBF3 subunits and for the CBF3-CDEIII DNA complex. (a) Representation of CBF3 subunits as ellipsoids (with circular cross-sections) whose longest axis is equal to twice the Stokes radius and whose shorter axis is calculated using the experimentally determined mass and a typical value for protein density. p64 is shown as having two domains. Interactions that have been detected among CBF3 subunits are shown with heavy arrows. X denotes those interactions that have not been be detected in solution. (b) Enlarged cross-section of the CBF3-DNA complex showing the interaction of p58 and p64 with conserved bases in CDEIII. The boxes mark bases that are absolutely conserved among all 16 yeast centromeres and arrows indicate the positions of the putative p64 half-sites. Cross-linking data are summarized from Espelin et al. (1997). Below the enlargement is the proposed arrangement of CBF3 subunits on CDEIII DNA. A 184-bp piece of DNA with an extended CBF3 complex is shown. For a sense of scale, the dotted circles show the approximate diameter of a nucleosome and a microtubule in cross-section. The dotted box indicates the region of the model used to generate the cross-sectional view shown in section c. A model for organization of the core CBF3-CDEIII complex on 56 bp of centromeric DNA. The complex contains one p58 subunit, one p23^Skp1, two p64s, and two p110s. The calculated mass of this complex (450 kD) and the experimentally determined sedimentation coefficient of 15 S yield a Stokes radius of 70 Å. CDEIII has been bent at an arbitrary position to conform to this 70 Å length. (d) Model for the regulation of CBF3 formation by p58. Newly synthesized p58 is activated by phosphorylation in a p23^Skp1-dependent manner. Both active and inactive p58 are subject to ubiquitination and subsequent degradation. p58 bound by p64 assumes a conformational state in which it is resistant to degradation. However, p64-bound p58 remains in equilibrium with the free p58. Association of the p58/p64 complex with p110 and centromeric DNA leads to the formation of a stable complex that dissociates only very slowly.

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