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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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Truncation of Rps5 46 N-terminal amino acid residues confers leaky scanning phenotype. Expression of reporter GCN4-lacZ constructs. rps5-Δ0 and isogenic rps5-Δ1-13, rps5-Δ1-24, rps5-Δ1-30, rps5-Δ1-46 strains were transformed with (A) p227 containing uORFs less GCN4 mRNA leader; (B) p209 containing uORF1; (C) p226 containing only uORF4 and (D) pM226 containing uORF1 extended into GCN4 ORF. β-Galactosidase activity (units) are shown; measured under normal (−SM) and aa starved conditions (+SM) as in Figure 1.
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Figure 2: Truncation of Rps5 46 N-terminal amino acid residues confers leaky scanning phenotype. Expression of reporter GCN4-lacZ constructs. rps5-Δ0 and isogenic rps5-Δ1-13, rps5-Δ1-24, rps5-Δ1-30, rps5-Δ1-46 strains were transformed with (A) p227 containing uORFs less GCN4 mRNA leader; (B) p209 containing uORF1; (C) p226 containing only uORF4 and (D) pM226 containing uORF1 extended into GCN4 ORF. β-Galactosidase activity (units) are shown; measured under normal (−SM) and aa starved conditions (+SM) as in Figure 1.

Mentions: To assess reinitiation in yeast strains expressing N-terminally truncated versions of Rps5, we assayed a set of GCN4-lacZ reporters with different arrangements of the uORFs (Figures 1 and 2 and Supplementary S1A). Plasmids were transformed into wild type strain rps5-Δ0 and its isogenic derivatives rps5-Δ1-13, rps5-Δ1-24, rps5-Δ1-30 and rps5-Δ1-46 carrying different truncated RPS5 genes. Cells were treated with sulfometuron methyl (SM), which inhibits isoleucine-valine biosynthesis, to derepress GCN4 expression (for review, see (26)). We found that truncation of 13 or 24 N-terminal amino acids in Rps5 does not substantially affect reinitiation in constructs containing all four uORFs (reporter plasmid p180, Figure 1A) or only uORFs 1 and 4 (p196, Figure 1B), as strains rps5-Δ1-13 and rps5-Δ1-24 exhibit basal levels of β-galactosidase activity under non-starvation conditions (−SM), and derepressed levels of activity under amino acid starvation conditions (+SM), that are nearly identical to those seen in the WT strain. Upstream ORFs 2 and 3 are known to be functionally redundant with uORF4 such that uORF1 and uORF4 are sufficient for nearly WT regulation of reinitiation on GCN4 mRNA (17). By contrast, GCN4-lacZ expression from p180 was reduced by ∼4-fold in rps5-Δ1-30 and by ∼40-fold in rps5-Δ1-46 in comparison with the WT strain (Figure 1A). Thus, while rps5-Δ1-30 retains a low-level induction of reinitiation (∼2-fold), both the basal level of reinitiation and its induction were almost completely abrogated in rps5-Δ1-46. The same holds true for the p196 construct that contains only uORFs 1 and 4 (Figure 1B). The GCN4-lacZ expression data were corroborated by growth assays (Figure 1C). Growth of rps5-Δ1-46 was compromised under amino acid starved conditions (Figure 1C, +SM), suggesting that GCN4 fails to efficiently activate the expression of genes encoding amino acid biosynthetic enzymes in this mutant (26). These observations led us to conclude that the rps5-Δ1-46 mutant exhibits a strong General control non-derepressible (Gcn−) phenotype (26).


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)

Truncation of Rps5 46 N-terminal amino acid residues confers leaky scanning phenotype. Expression of reporter GCN4-lacZ constructs. rps5-Δ0 and isogenic rps5-Δ1-13, rps5-Δ1-24, rps5-Δ1-30, rps5-Δ1-46 strains were transformed with (A) p227 containing uORFs less GCN4 mRNA leader; (B) p209 containing uORF1; (C) p226 containing only uORF4 and (D) pM226 containing uORF1 extended into GCN4 ORF. β-Galactosidase activity (units) are shown; measured under normal (−SM) and aa starved conditions (+SM) as in Figure 1.
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Related In: Results  -  Collection

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Figure 2: Truncation of Rps5 46 N-terminal amino acid residues confers leaky scanning phenotype. Expression of reporter GCN4-lacZ constructs. rps5-Δ0 and isogenic rps5-Δ1-13, rps5-Δ1-24, rps5-Δ1-30, rps5-Δ1-46 strains were transformed with (A) p227 containing uORFs less GCN4 mRNA leader; (B) p209 containing uORF1; (C) p226 containing only uORF4 and (D) pM226 containing uORF1 extended into GCN4 ORF. β-Galactosidase activity (units) are shown; measured under normal (−SM) and aa starved conditions (+SM) as in Figure 1.
Mentions: To assess reinitiation in yeast strains expressing N-terminally truncated versions of Rps5, we assayed a set of GCN4-lacZ reporters with different arrangements of the uORFs (Figures 1 and 2 and Supplementary S1A). Plasmids were transformed into wild type strain rps5-Δ0 and its isogenic derivatives rps5-Δ1-13, rps5-Δ1-24, rps5-Δ1-30 and rps5-Δ1-46 carrying different truncated RPS5 genes. Cells were treated with sulfometuron methyl (SM), which inhibits isoleucine-valine biosynthesis, to derepress GCN4 expression (for review, see (26)). We found that truncation of 13 or 24 N-terminal amino acids in Rps5 does not substantially affect reinitiation in constructs containing all four uORFs (reporter plasmid p180, Figure 1A) or only uORFs 1 and 4 (p196, Figure 1B), as strains rps5-Δ1-13 and rps5-Δ1-24 exhibit basal levels of β-galactosidase activity under non-starvation conditions (−SM), and derepressed levels of activity under amino acid starvation conditions (+SM), that are nearly identical to those seen in the WT strain. Upstream ORFs 2 and 3 are known to be functionally redundant with uORF4 such that uORF1 and uORF4 are sufficient for nearly WT regulation of reinitiation on GCN4 mRNA (17). By contrast, GCN4-lacZ expression from p180 was reduced by ∼4-fold in rps5-Δ1-30 and by ∼40-fold in rps5-Δ1-46 in comparison with the WT strain (Figure 1A). Thus, while rps5-Δ1-30 retains a low-level induction of reinitiation (∼2-fold), both the basal level of reinitiation and its induction were almost completely abrogated in rps5-Δ1-46. The same holds true for the p196 construct that contains only uORFs 1 and 4 (Figure 1B). The GCN4-lacZ expression data were corroborated by growth assays (Figure 1C). Growth of rps5-Δ1-46 was compromised under amino acid starved conditions (Figure 1C, +SM), suggesting that GCN4 fails to efficiently activate the expression of genes encoding amino acid biosynthetic enzymes in this mutant (26). These observations led us to conclude that the rps5-Δ1-46 mutant exhibits a strong General control non-derepressible (Gcn−) phenotype (26).

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