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. 2019 Jun 21;10:2754. doi: 10.1038/s41467-019-10678-z

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — A genetic network between Rps20 loop and 40S maturation mutants. a–c rps20 loop alleles enhance the phenotypes of mutants that impair Rps3 N-domain assembly. a An RPS20 (rps20Δ) shuffle strain was transformed with the indicated plasmid-based rps20 alleles. Transformants were spotted in ten-fold serial dilutions on SDC-Leu and SDC+5-FOA plates and growth at 30 °C was monitored after 3 and 5 days, respectively. b An RPS20 (rps20Δ) RPS3 (rps3Δ) double shuffle strain was transformed with the indicated plasmid-based RPS20 and RPS3 wild-type and mutant alleles. Transformants were spotted in ten-fold serial dilutions on SDC-Leu-Trp and SDC+5-FOA plates and growth at 30 °C was monitored after 3 and 5 days, respectively. c Polysome profiles of cells expressing the indicated rps20 alleles, revealing an increased 40S synthesis defect when a rps20 loop mutation is combined with a rps20 allele that affects the interaction with the Rps3 N-domain (R68/K69>A D113/E115>K mutant). The panels below the profiles show growth of the respective strains on YPD plates incubated for 2 days at 30 °C. d, e Genetic interactions between rps20 loop alleles and mutants of pre-40S assembly factors (AFs). An RPS20 (rps20Δ) RIO2 (rio2Δ) double shuffle strain (d) and an RPS20 (rps20Δ) shuffle ltv1Δ strain (e) were transformed with the indicated plasmid-based alleles. Transformants were spotted in ten-fold serial dilutions on SDC-Leu-Trp (-leu-trp) or 5-FOA-containing medium and growth at 30 °C was monitored at the indicated days. f Genetic network illustrating the interplay of the Rps20 loop with AFs and r-proteins at distant sites on the pre-40S ribosome. All genetic interactions discovered in this study (Figs. 2 and 3) and in our previous study¹⁹ are indicated by dashed lines. g The Rps20 loop is required for AF release from cytoplasmic pre-40S particles. Tsr1-TAP particles were purified from wild-type RPS20 or rps20Δloop yeast strains. Eluates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting using the indicated specific antibodies