Sucrose gradient analysis of ribosomes after repressing the genes for 60S r-proteins uL4 and uL22 and 60S assembly factors Rpf2 and Rrs1. Sucrose gradient.

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Sucrose gradient analysis of ribosomes after repressing the genes for 60S r-proteins uL4 and uL22 and 60S assembly factors Rpf2 and Rrs1. Sucrose gradient analysis of ribosomes after repressing the genes for 60S r-proteins uL4 and uL22 and 60S assembly factors Rpf2 and Rrs1. Cultures were grown in YEP-galactose medium and shifted to YPD glucose medium for the indicated lengths of time before harvest. (A, B) The control strain BY4741 in galactose and glucose medium, respectively. (C, D) Pgal-uL4 at 0 and 8 h, respectively. (E–H) Pgal-uL22 at 0, 2.5, 8, and 16 h, respectively. (I, J) Pgal-Rpf2 at 0 and 16 h, respectively. (K, L) Pgal-Rrs1 at 0 and 16 h, respectively. For C and D, equal volumes of fractions in the ribosome portion of the gradient were analyzed for r-proteins uS4, uL4, uL5, and uL18 by Western blot. (M, N) Quantification of 40S subunits. The area under each ribosomal peek was measured using ImageJ and normalized to the area under all ribosomal peaks. (M) The fraction of ribosomal mass in 40S subunits was calculated as the fraction found in the free 40S peak plus 1/3 of the fraction in 80S and polysomes. (N) The fraction of ribosomal mass in free 40S subunits. Red and green circles refer to repression of uL22 and uL4 synthesis, respectively. Purple and blue triangles refer to repression of Rpf2 and Rrs1 synthesis, respectively. Brian Gregory et al. LSA 2019;2:e201800150 © 2019 Gregory et al.