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A conserved and essential basic region mediates tRNA binding to the Elp1 subunit of the Saccharomyces cerevisiae Elongator complex.

Di Santo R, Bandau S, Stark MJ - Mol. Microbiol. (2014)

Bottom Line: Since these modifications are required for the tRNAs to function efficiently, a translation defect caused by hypomodified tRNAs may therefore underlie the variety of phenotypes associated with Elongator dysfunction.The Elp1 carboxy-terminal domain contains a highly conserved arginine/lysine-rich region that resembles a nuclear localization sequence (NLS).Thus the conserved basic region in Elp1 may be essential for tRNA wobble uridine modification by acting as tRNA binding motif.

View Article: PubMed Central - PubMed

Affiliation: Centre for Gene Regulation & Expression, College of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, DD1 5EH, Scotland, UK.

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Related in: MedlinePlus

A highly conserved basic region in Elp1 is essential for Elongator function.A. Multiple sequence alignment of the Elp1 subunit of Elongator from various eukaryotic organisms showing a portion of the Elp1 C-terminal domain including the basic region to highlight its conservation. Alignment was carried out using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Sequences used were as follows with accession numbers in brackets; S. cerevisiae (Q06706), S. pombe (O59704), H. sapiens (O95163), M. musculus (Q7TT37), O. cuniculus (Q8WND5), R. norveigus (Q8VHU4), X. laevis (Q2TAQ1), D. melanogaster (Q9VGK7), A. thaliana (Q9FNA4).B. Schematic of Elp1 highlighting the Arg/Lys rich basic region (basic residues in bold) and alanine substitution mutations (boxed) made within this region. Mutations were made across the entire Elp1 basic region (elp1–KR9A) and within the first and second halves of the region (elp1–KR5A and elp1–KR4A respectively). For comparison the consensus sequence for a bipartite NLS is also shown. The indicated structural predictions were made using PSIPRED (Buchan et al., 2013).C. Zymocin sensitivity assays of strains containing GFP tagged wild-type ELP1 or elp1 basic region mutants at the ELP1 genomic locus. Wild-type (YRDS253) and elp1Δ (YRDS250) served as controls for zymocin sensitivity and resistance respectively. Left panel, Eclipse assays on YPAD medium measuring growth inhibition adjacent to a colony of a zymocin-secreting K. lactis strain. Centre and right panels, growth of equivalent 10-fold serial dilutions (indicating OD600) of the indicated strains containing pAE1, which expresses the zymocin γ subunit under control of a galactose inducible promoter. Strains were grown on YPARG (centre panel: zymocin induced) and YPAD (right panel: zymocin uninduced).D. Growth of equivalent 10-fold serial dilutions of the indicated strains containing chromosomal copies of ELP1 or elp1–KR9A together with the SUP4 ochre suppressor and either wild-type URA3 (as a control) or the ura3oc22 ochre allele. SUP4 can read through the premature stop codon in ura3oc22 to allow growth in the absence of uracil on DOA-Ura medium but efficient suppression requires mcm5 modification of the SUP4 tRNA at the wobble uridine position, as shown by comparing growth of the ELP1 and elp1Δ ura3oc22 strains. Growth of the same strains on synthetic complete (SC) medium without uracil selection is shown as a control.
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fig01: A highly conserved basic region in Elp1 is essential for Elongator function.A. Multiple sequence alignment of the Elp1 subunit of Elongator from various eukaryotic organisms showing a portion of the Elp1 C-terminal domain including the basic region to highlight its conservation. Alignment was carried out using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Sequences used were as follows with accession numbers in brackets; S. cerevisiae (Q06706), S. pombe (O59704), H. sapiens (O95163), M. musculus (Q7TT37), O. cuniculus (Q8WND5), R. norveigus (Q8VHU4), X. laevis (Q2TAQ1), D. melanogaster (Q9VGK7), A. thaliana (Q9FNA4).B. Schematic of Elp1 highlighting the Arg/Lys rich basic region (basic residues in bold) and alanine substitution mutations (boxed) made within this region. Mutations were made across the entire Elp1 basic region (elp1–KR9A) and within the first and second halves of the region (elp1–KR5A and elp1–KR4A respectively). For comparison the consensus sequence for a bipartite NLS is also shown. The indicated structural predictions were made using PSIPRED (Buchan et al., 2013).C. Zymocin sensitivity assays of strains containing GFP tagged wild-type ELP1 or elp1 basic region mutants at the ELP1 genomic locus. Wild-type (YRDS253) and elp1Δ (YRDS250) served as controls for zymocin sensitivity and resistance respectively. Left panel, Eclipse assays on YPAD medium measuring growth inhibition adjacent to a colony of a zymocin-secreting K. lactis strain. Centre and right panels, growth of equivalent 10-fold serial dilutions (indicating OD600) of the indicated strains containing pAE1, which expresses the zymocin γ subunit under control of a galactose inducible promoter. Strains were grown on YPARG (centre panel: zymocin induced) and YPAD (right panel: zymocin uninduced).D. Growth of equivalent 10-fold serial dilutions of the indicated strains containing chromosomal copies of ELP1 or elp1–KR9A together with the SUP4 ochre suppressor and either wild-type URA3 (as a control) or the ura3oc22 ochre allele. SUP4 can read through the premature stop codon in ura3oc22 to allow growth in the absence of uracil on DOA-Ura medium but efficient suppression requires mcm5 modification of the SUP4 tRNA at the wobble uridine position, as shown by comparing growth of the ELP1 and elp1Δ ura3oc22 strains. Growth of the same strains on synthetic complete (SC) medium without uracil selection is shown as a control.

Mentions: The yeast Elp1 C-terminal domain contains an Arg/Lys-rich basic region that is highly conserved across eukaryotes (Fig. 1A), implying that it could be functionally important. This region had previously been noted to resemble a putative bipartite NLS (Fichtner et al., 2003), which has the classical consensus sequence (K/R)(K/R)X10–12(K/R)3/5 (Lange et al., 2007; Kosugi et al., 2009). To study the functional significance of this conserved basic region a series of mutations were designed throughout its sequence. A mutant allele (elp1–KR9A) was generated in which nine of the basic residues were substituted by alanine, as well as elp1–KR5A and elp1–KR4A alleles containing alanine substitutions in the first and second halves of the basic region respectively (Fig. 1B). All three mutants were incorporated into the genomic ELP1 locus using the ‘delitto perfetto’ approach.


A conserved and essential basic region mediates tRNA binding to the Elp1 subunit of the Saccharomyces cerevisiae Elongator complex.

Di Santo R, Bandau S, Stark MJ - Mol. Microbiol. (2014)

A highly conserved basic region in Elp1 is essential for Elongator function.A. Multiple sequence alignment of the Elp1 subunit of Elongator from various eukaryotic organisms showing a portion of the Elp1 C-terminal domain including the basic region to highlight its conservation. Alignment was carried out using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Sequences used were as follows with accession numbers in brackets; S. cerevisiae (Q06706), S. pombe (O59704), H. sapiens (O95163), M. musculus (Q7TT37), O. cuniculus (Q8WND5), R. norveigus (Q8VHU4), X. laevis (Q2TAQ1), D. melanogaster (Q9VGK7), A. thaliana (Q9FNA4).B. Schematic of Elp1 highlighting the Arg/Lys rich basic region (basic residues in bold) and alanine substitution mutations (boxed) made within this region. Mutations were made across the entire Elp1 basic region (elp1–KR9A) and within the first and second halves of the region (elp1–KR5A and elp1–KR4A respectively). For comparison the consensus sequence for a bipartite NLS is also shown. The indicated structural predictions were made using PSIPRED (Buchan et al., 2013).C. Zymocin sensitivity assays of strains containing GFP tagged wild-type ELP1 or elp1 basic region mutants at the ELP1 genomic locus. Wild-type (YRDS253) and elp1Δ (YRDS250) served as controls for zymocin sensitivity and resistance respectively. Left panel, Eclipse assays on YPAD medium measuring growth inhibition adjacent to a colony of a zymocin-secreting K. lactis strain. Centre and right panels, growth of equivalent 10-fold serial dilutions (indicating OD600) of the indicated strains containing pAE1, which expresses the zymocin γ subunit under control of a galactose inducible promoter. Strains were grown on YPARG (centre panel: zymocin induced) and YPAD (right panel: zymocin uninduced).D. Growth of equivalent 10-fold serial dilutions of the indicated strains containing chromosomal copies of ELP1 or elp1–KR9A together with the SUP4 ochre suppressor and either wild-type URA3 (as a control) or the ura3oc22 ochre allele. SUP4 can read through the premature stop codon in ura3oc22 to allow growth in the absence of uracil on DOA-Ura medium but efficient suppression requires mcm5 modification of the SUP4 tRNA at the wobble uridine position, as shown by comparing growth of the ELP1 and elp1Δ ura3oc22 strains. Growth of the same strains on synthetic complete (SC) medium without uracil selection is shown as a control.
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fig01: A highly conserved basic region in Elp1 is essential for Elongator function.A. Multiple sequence alignment of the Elp1 subunit of Elongator from various eukaryotic organisms showing a portion of the Elp1 C-terminal domain including the basic region to highlight its conservation. Alignment was carried out using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Sequences used were as follows with accession numbers in brackets; S. cerevisiae (Q06706), S. pombe (O59704), H. sapiens (O95163), M. musculus (Q7TT37), O. cuniculus (Q8WND5), R. norveigus (Q8VHU4), X. laevis (Q2TAQ1), D. melanogaster (Q9VGK7), A. thaliana (Q9FNA4).B. Schematic of Elp1 highlighting the Arg/Lys rich basic region (basic residues in bold) and alanine substitution mutations (boxed) made within this region. Mutations were made across the entire Elp1 basic region (elp1–KR9A) and within the first and second halves of the region (elp1–KR5A and elp1–KR4A respectively). For comparison the consensus sequence for a bipartite NLS is also shown. The indicated structural predictions were made using PSIPRED (Buchan et al., 2013).C. Zymocin sensitivity assays of strains containing GFP tagged wild-type ELP1 or elp1 basic region mutants at the ELP1 genomic locus. Wild-type (YRDS253) and elp1Δ (YRDS250) served as controls for zymocin sensitivity and resistance respectively. Left panel, Eclipse assays on YPAD medium measuring growth inhibition adjacent to a colony of a zymocin-secreting K. lactis strain. Centre and right panels, growth of equivalent 10-fold serial dilutions (indicating OD600) of the indicated strains containing pAE1, which expresses the zymocin γ subunit under control of a galactose inducible promoter. Strains were grown on YPARG (centre panel: zymocin induced) and YPAD (right panel: zymocin uninduced).D. Growth of equivalent 10-fold serial dilutions of the indicated strains containing chromosomal copies of ELP1 or elp1–KR9A together with the SUP4 ochre suppressor and either wild-type URA3 (as a control) or the ura3oc22 ochre allele. SUP4 can read through the premature stop codon in ura3oc22 to allow growth in the absence of uracil on DOA-Ura medium but efficient suppression requires mcm5 modification of the SUP4 tRNA at the wobble uridine position, as shown by comparing growth of the ELP1 and elp1Δ ura3oc22 strains. Growth of the same strains on synthetic complete (SC) medium without uracil selection is shown as a control.
Mentions: The yeast Elp1 C-terminal domain contains an Arg/Lys-rich basic region that is highly conserved across eukaryotes (Fig. 1A), implying that it could be functionally important. This region had previously been noted to resemble a putative bipartite NLS (Fichtner et al., 2003), which has the classical consensus sequence (K/R)(K/R)X10–12(K/R)3/5 (Lange et al., 2007; Kosugi et al., 2009). To study the functional significance of this conserved basic region a series of mutations were designed throughout its sequence. A mutant allele (elp1–KR9A) was generated in which nine of the basic residues were substituted by alanine, as well as elp1–KR5A and elp1–KR4A alleles containing alanine substitutions in the first and second halves of the basic region respectively (Fig. 1B). All three mutants were incorporated into the genomic ELP1 locus using the ‘delitto perfetto’ approach.

Bottom Line: Since these modifications are required for the tRNAs to function efficiently, a translation defect caused by hypomodified tRNAs may therefore underlie the variety of phenotypes associated with Elongator dysfunction.The Elp1 carboxy-terminal domain contains a highly conserved arginine/lysine-rich region that resembles a nuclear localization sequence (NLS).Thus the conserved basic region in Elp1 may be essential for tRNA wobble uridine modification by acting as tRNA binding motif.

View Article: PubMed Central - PubMed

Affiliation: Centre for Gene Regulation & Expression, College of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, DD1 5EH, Scotland, UK.

Show MeSH
Related in: MedlinePlus