FIG 1.

The deletion of the yeast P1α-P2β stalk dimer displays a general control phenotype. (A) Table summarizing strains used, their genotypes, stalk proteins expressed, 60S/40S abundance ratios, and 60S abundance levels computed as the ratio of the 60S/40S abundance compared to that of the wild type. The 60S/40S abundance ratio was measured by growing the indicated strains in a complex medium, yeast extract-peptone-dextrose (YPD), and analyzing ribosomes in their lysates through density gradient-velocity sedimentation, as described in Materials and Methods. The averages of data from two experiments are shown with standard deviations. (B) Model of GCN4 translational control and its deregulation by 60S subunit mutations. The schematics at the top describe the structure of GCN4 mRNA, with boxes indicating uORFs. The table below describes the uORF-dependent delayed-reinitiation model for GCN4 translation in the wild type (rows 1 and 2) or eIF5B (6) or 60S (8, 26, 27) mutants (rows 3 to 5). Ovals in the schematics represent ribosomes with 40S and 60S subunits or the subunit alone. Brown arrows indicate ribosome dissociation. See the text for details. For the vertical arrow, we propose that distinct general control phenotypes observed with 60S subunit mutants can be explained by a different severity of subunit-joining defects resulting in a corresponding degree of leaky scanning of start codons (see also Fig. S1 and the text in the supplemental material). (C) The transformants of the indicated strains carrying the GCN4-lacZ plasmid, p180, and an empty vector (to match growth media with experiments in Fig. 2C) were grown at 30°C in the absence (3AT−) (bars 1, 3, and 5) or presence (3AT+) (bars 2, 4, and 6) of 10 mM 3AT for 6 h, following preculturing for 2 h, and then assayed for β-galactosidase (β-gal) activity, as described in Materials and Methods. β-Galactosidase activity, in Miller units, is presented as averages and standard errors of six values obtained in duplicate from 3 independent experiments. Gcd, general control derepressed; Gcn, general control nonderepressible. Data for D57 were adapted from data shown in Fig. 2C, bars 3 and 4. (D and E) Complementation of phenotypes in D57 by P1α-P2β. Transformants of the indicated stains carrying empty vector (Vec) or YCpU-RPP1A and YCpW-RPP2B (P1α:P2β) were grown in synthetic complete (SC) medium lacking uracil and tryptophan and subjected to 3AT sensitivity (D) and ribosome subunit abundance (E) assays. (D) Fixed amounts (5 μl of culture diluted to an optical density at 600 nm [OD₆₀₀] of 0.15) of the indicated transformants and their 10-fold serial dilutions were spotted onto SC medium plates lacking uracil, tryptophan, and histidine supplemented with 40 mM leucine with (+3AT) or without (−3AT) 40 mM 3AT and grown for 4 days at 30°C. Colonies are reddish due to the ade2 marker. (E) A₂₅₄ profiles of ribosome subunit abundances are shown with 60S/40S ratios (n = 2 with standard deviations).