Fig. 2.

Binding of Mpy requires Mrf-associated ribosomes. (a) Immunoblot analysis of the presence of Mpy in ribosomes purified from a mixture containing recombinant Mpy (rMpy) and crude ribosomes from indicated strains (in parenthesis) grown under either low-zinc (1 µM TPEN) or high-zinc (1 mM ZnSO4) Sauton’s medium for 4 days. The type of ribosomes obtained from each strain under each growth condition is indicated above the lane as C+ or C-. The strain pYL53 constitutively expressed C- ribosomes, even under high-zinc conditions. Crude ribosomes equivalent to OD 0.1 were mixed with 2.4 picomoles of rMpy in a 50 µl reaction and the mixture was incubated at 37 ^∘C for 1 h. Then the ribosomes were separated from the unbound rMPY by ultracentrifugation of the mixture on a 32 % sucrose cushion at 100 000 r.p.m. for 3 h in Beckman TLA 100.3 rotor. The pellets containing the ribosomes were analysed for the presence of rMpy. S13 was probed as the loading control. (b) Quantitative difference between the level of rMpy in the indicated ribosome samples, relative to low-zinc C- ribosomes (normalized as 1) obtained from immunoblots including the one shown in (a). Data represent the average of two independent binding experiments from biologically independent preparations of crude ribosomes and rMPy. * and ** represent P-value (t-test) <0.05 and 0.01, respectively.