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Rps5-Rps16 communication is essential for efficient translation initiation in yeast S. cerevisiae.

Ghosh A, Jindal S, Bentley AA, Hinnebusch AG, Komar AA - Nucleic Acids Res. (2014)

Bottom Line: Rps5 mutations evoke accumulation of factors on native 40S subunits normally released on conversion of 48S PICs to 80S initiation complexes (ICs) and this abnormality and related phenotypes are mitigated by the SUI5 variant of eIF5.Remarkably, similar effects are observed by substitution of Lys45 in the Rps5-NTD, involved in contact with Rps16, and by eliminating the last two residues of the C-terminal tail (CTT) of Rps16, believed to contact initiator tRNA base-paired to AUG in the P site.We propose that Rps5-NTD-Rps16-NTD interaction modulates Rps16-CTT association with Met-tRNAi (Met) to promote a functional 48S PIC.

View Article: PubMed Central - PubMed

Affiliation: Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, OH 44115, USA.

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A single point K45A mutation in Rps5 confers phenotype as in rps5-Δ1-46 strain. (A) Yeast cell growth. Serial dilutions of yeast strains with the indicated genotypes (harboring point mutation in Rps5; K41A, F43G and K45A) and rps5-Δ1-46 were spotted onto YPD agar plates with 2% glucose. (B) Ribosome profiles of the wt, rps5-Δ1-46 and rps5-K45A mutant yeast strains. Extracts were resolved in 10–50% sucrose density gradients. The ratios of the area under the polysomal (P) and 80S (M) peaks (P:M) are shown with ± standard errors. rps5-Δ1-46 and rps5-K45A mutant yeast strains reveal very similar ribosome profiles. (C) rps5-Δ0 and rps5-K45A yeast strains were transformed with reporter construct p180 containing wild type GCN4 mRNA leader (with all four uORFs) upstream of GCN4-LacZ fusion gene. The strains were assayed for β-galactosidase activity under normal, nutrient rich, (−SM) and aa starved (+SM) conditions as in Figure 1.
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Figure 6: A single point K45A mutation in Rps5 confers phenotype as in rps5-Δ1-46 strain. (A) Yeast cell growth. Serial dilutions of yeast strains with the indicated genotypes (harboring point mutation in Rps5; K41A, F43G and K45A) and rps5-Δ1-46 were spotted onto YPD agar plates with 2% glucose. (B) Ribosome profiles of the wt, rps5-Δ1-46 and rps5-K45A mutant yeast strains. Extracts were resolved in 10–50% sucrose density gradients. The ratios of the area under the polysomal (P) and 80S (M) peaks (P:M) are shown with ± standard errors. rps5-Δ1-46 and rps5-K45A mutant yeast strains reveal very similar ribosome profiles. (C) rps5-Δ0 and rps5-K45A yeast strains were transformed with reporter construct p180 containing wild type GCN4 mRNA leader (with all four uORFs) upstream of GCN4-LacZ fusion gene. The strains were assayed for β-galactosidase activity under normal, nutrient rich, (−SM) and aa starved (+SM) conditions as in Figure 1.

Mentions: To test this possibility, we mutated conserved Rps5 residues K41, F43 and K45, substituting positively charged K41 and K45 with alanines and bulky aromatic F43 with glycine and obtained yeast strains in which the wild type yeast RPS5 was replaced by the mutant RPS5 alleles. Interestingly, rps5-K41A (harboring lysine to alanine substitution at Rps5 position 41) and rps5-F43G (harboring phenylalanine to glycine substitution at position 43) exhibited growth rates very similar to that of the wild type strain, while rps5-K45A displayed a pronounced Slg− phenotype approaching that of the rps5-Δ1-46 strain (Figure 6A). Analysis of polysomes by sedimentation of whole cell extracts through sucrose density-gradients further revealed a marked reduction in polysome (P) to monosome (80S) ratio (P:M) in the rps5-K45A mutant as compared to the wild type strain, albeit not as severe as that seen for the rps5-Δ1-46 mutant (Figure 6B). A decrease in the P:M ratio is a characteristic phenotype of mutations that impair translation initiation without a commensurate reduction in the elongation or termination phases of translation, reducing the average number of ribosomes per mRNA. Importantly, we also found that the rps5-K45A mutant resembles the rps5-Δ1-46 strain in displaying a Gcn− phenotype, as indicated by increased sensitivity to SM (Supplementary Figure S4A) and reduced β-galactosidase activity expressed from the GCN4-lacZ reporter on p180 (Figure 6C and Supplementary Figure S4B); and also in displaying a reduced UUG:AUG ratio for the HIS4-lacZ reporters (Figure 3C and Supplementary Figure S3). Like the Slg− phenotype and reduction in P:M ratio (Figure 6A and B), both defects were less pronounced in rps5-K45A versus the rps5-Δ1-46 mutant. Interestingly, the reciprocal F46A mutation in RPS16 confers a slow growth phenotype (Supplementary Figure S5A) similar in degree to that observed for the rps5-K45A mutant (Figure 6A). In addition, Rps16 Tyrosine 49 also forms contacts with the Rps5 N-terminus, and the Y49G mutation in RPS16 (4,5) confers an even stronger Slg− phenotype (Supplementary Figure S5A). Importantly, F46A mutation causes a similar reduction in polysome (P) to monosome (80S) ratio (P:M) (as in the rps5-K45A mutant) as compared to the wild RPS16 type strain (Supplementary Figure S5B). Although further experiments are required to determine the exact consequences of these RPS16 mutations, these data support the possibility that interaction between the N-terminal regions of Rps5 and Rps16 is critical for efficient translation initiation at a step following AUG recognition. This further raises the question of how structural changes on the solvent side of the 40S ribosomal subunit may affect TC recruitment and GTP hydrolysis.


Rps5-Rps16 communication is essential for efficient translation initiation in yeast S. cerevisiae.

Ghosh A, Jindal S, Bentley AA, Hinnebusch AG, Komar AA - Nucleic Acids Res. (2014)

A single point K45A mutation in Rps5 confers phenotype as in rps5-Δ1-46 strain. (A) Yeast cell growth. Serial dilutions of yeast strains with the indicated genotypes (harboring point mutation in Rps5; K41A, F43G and K45A) and rps5-Δ1-46 were spotted onto YPD agar plates with 2% glucose. (B) Ribosome profiles of the wt, rps5-Δ1-46 and rps5-K45A mutant yeast strains. Extracts were resolved in 10–50% sucrose density gradients. The ratios of the area under the polysomal (P) and 80S (M) peaks (P:M) are shown with ± standard errors. rps5-Δ1-46 and rps5-K45A mutant yeast strains reveal very similar ribosome profiles. (C) rps5-Δ0 and rps5-K45A yeast strains were transformed with reporter construct p180 containing wild type GCN4 mRNA leader (with all four uORFs) upstream of GCN4-LacZ fusion gene. The strains were assayed for β-galactosidase activity under normal, nutrient rich, (−SM) and aa starved (+SM) conditions as in Figure 1.
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Figure 6: A single point K45A mutation in Rps5 confers phenotype as in rps5-Δ1-46 strain. (A) Yeast cell growth. Serial dilutions of yeast strains with the indicated genotypes (harboring point mutation in Rps5; K41A, F43G and K45A) and rps5-Δ1-46 were spotted onto YPD agar plates with 2% glucose. (B) Ribosome profiles of the wt, rps5-Δ1-46 and rps5-K45A mutant yeast strains. Extracts were resolved in 10–50% sucrose density gradients. The ratios of the area under the polysomal (P) and 80S (M) peaks (P:M) are shown with ± standard errors. rps5-Δ1-46 and rps5-K45A mutant yeast strains reveal very similar ribosome profiles. (C) rps5-Δ0 and rps5-K45A yeast strains were transformed with reporter construct p180 containing wild type GCN4 mRNA leader (with all four uORFs) upstream of GCN4-LacZ fusion gene. The strains were assayed for β-galactosidase activity under normal, nutrient rich, (−SM) and aa starved (+SM) conditions as in Figure 1.
Mentions: To test this possibility, we mutated conserved Rps5 residues K41, F43 and K45, substituting positively charged K41 and K45 with alanines and bulky aromatic F43 with glycine and obtained yeast strains in which the wild type yeast RPS5 was replaced by the mutant RPS5 alleles. Interestingly, rps5-K41A (harboring lysine to alanine substitution at Rps5 position 41) and rps5-F43G (harboring phenylalanine to glycine substitution at position 43) exhibited growth rates very similar to that of the wild type strain, while rps5-K45A displayed a pronounced Slg− phenotype approaching that of the rps5-Δ1-46 strain (Figure 6A). Analysis of polysomes by sedimentation of whole cell extracts through sucrose density-gradients further revealed a marked reduction in polysome (P) to monosome (80S) ratio (P:M) in the rps5-K45A mutant as compared to the wild type strain, albeit not as severe as that seen for the rps5-Δ1-46 mutant (Figure 6B). A decrease in the P:M ratio is a characteristic phenotype of mutations that impair translation initiation without a commensurate reduction in the elongation or termination phases of translation, reducing the average number of ribosomes per mRNA. Importantly, we also found that the rps5-K45A mutant resembles the rps5-Δ1-46 strain in displaying a Gcn− phenotype, as indicated by increased sensitivity to SM (Supplementary Figure S4A) and reduced β-galactosidase activity expressed from the GCN4-lacZ reporter on p180 (Figure 6C and Supplementary Figure S4B); and also in displaying a reduced UUG:AUG ratio for the HIS4-lacZ reporters (Figure 3C and Supplementary Figure S3). Like the Slg− phenotype and reduction in P:M ratio (Figure 6A and B), both defects were less pronounced in rps5-K45A versus the rps5-Δ1-46 mutant. Interestingly, the reciprocal F46A mutation in RPS16 confers a slow growth phenotype (Supplementary Figure S5A) similar in degree to that observed for the rps5-K45A mutant (Figure 6A). In addition, Rps16 Tyrosine 49 also forms contacts with the Rps5 N-terminus, and the Y49G mutation in RPS16 (4,5) confers an even stronger Slg− phenotype (Supplementary Figure S5A). Importantly, F46A mutation causes a similar reduction in polysome (P) to monosome (80S) ratio (P:M) (as in the rps5-K45A mutant) as compared to the wild RPS16 type strain (Supplementary Figure S5B). Although further experiments are required to determine the exact consequences of these RPS16 mutations, these data support the possibility that interaction between the N-terminal regions of Rps5 and Rps16 is critical for efficient translation initiation at a step following AUG recognition. This further raises the question of how structural changes on the solvent side of the 40S ribosomal subunit may affect TC recruitment and GTP hydrolysis.

Bottom Line: Rps5 mutations evoke accumulation of factors on native 40S subunits normally released on conversion of 48S PICs to 80S initiation complexes (ICs) and this abnormality and related phenotypes are mitigated by the SUI5 variant of eIF5.Remarkably, similar effects are observed by substitution of Lys45 in the Rps5-NTD, involved in contact with Rps16, and by eliminating the last two residues of the C-terminal tail (CTT) of Rps16, believed to contact initiator tRNA base-paired to AUG in the P site.We propose that Rps5-NTD-Rps16-NTD interaction modulates Rps16-CTT association with Met-tRNAi (Met) to promote a functional 48S PIC.

View Article: PubMed Central - PubMed

Affiliation: Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, OH 44115, USA.

Show MeSH
Related in: MedlinePlus