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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

The Elp1 basic region is predicted to bind RNA and is required for formation of a complex with yeast tRNA.A. Analysis for predicted RNA binding sites within the S. cerevisiae and H. sapiens Elp1 protein sequence using BindN+ (http://bioinfo.ggc.org/bindn+/) showing the high confidence prediction of the basic region as a putative RNA binding region (confidence is scored on a scale of 1–9 with 9 as highest confidence). The locations of the Elp1 basic region and the elp1–KR9A alanine substitution mutations are shown for comparison. Overall conservation between the human and yeast sequences is also indicated (/, identity; •, similar residues).B. Recombinant Elp1 and Elp1–KR9A C-terminal domains (CTDs). Elp1 CTDs with N-terminal GST and C-terminal His6 tags were expressed and purified from E. coli along with GST-His6 as a control. The corresponding proteins were analysed on a 4–12% polyacrylamide gel and stained with instant blue. The GST–Elp1–CTD-His6 fusion proteins are indicated by an arrow.C. Electrophoretic mobility shift assay (EMSA) using ∼ 1 nM 32P-labelled yeast tRNA and the indicated concentrations of recombinant GST, GST–Elp1 and GST–Elp1–KR9A fusion proteins shown in (B). Binding reactions were separated on a 1.5% native agarose gel and analysed by autoradiography. A complex was formed between tRNA and the wild-type Elp1 CTD but not with the Elp1–KR9A CTD.
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fig06: The Elp1 basic region is predicted to bind RNA and is required for formation of a complex with yeast tRNA.A. Analysis for predicted RNA binding sites within the S. cerevisiae and H. sapiens Elp1 protein sequence using BindN+ (http://bioinfo.ggc.org/bindn+/) showing the high confidence prediction of the basic region as a putative RNA binding region (confidence is scored on a scale of 1–9 with 9 as highest confidence). The locations of the Elp1 basic region and the elp1–KR9A alanine substitution mutations are shown for comparison. Overall conservation between the human and yeast sequences is also indicated (/, identity; •, similar residues).B. Recombinant Elp1 and Elp1–KR9A C-terminal domains (CTDs). Elp1 CTDs with N-terminal GST and C-terminal His6 tags were expressed and purified from E. coli along with GST-His6 as a control. The corresponding proteins were analysed on a 4–12% polyacrylamide gel and stained with instant blue. The GST–Elp1–CTD-His6 fusion proteins are indicated by an arrow.C. Electrophoretic mobility shift assay (EMSA) using ∼ 1 nM 32P-labelled yeast tRNA and the indicated concentrations of recombinant GST, GST–Elp1 and GST–Elp1–KR9A fusion proteins shown in (B). Binding reactions were separated on a 1.5% native agarose gel and analysed by autoradiography. A complex was formed between tRNA and the wild-type Elp1 CTD but not with the Elp1–KR9A CTD.

Mentions: The conserved basic region in Elp1 is essential for tRNA wobble uridine modification but despite being capable of functioning as an NLS, it does not appear to influence the distribution of the Elp1 between the nucleus and cytoplasm and is not required for overall assembly of the Elongator complex. In keeping with Elongator's role in tRNA wobble uridine modification, it was recently shown by electrophoretic mobility shift assay (EMSA) that the Elp4–6 hexameric subcomplex of Elongator directly binds tRNAGlu(UUC), consistent with a direct role for Elongator in tRNA binding and subsequent modification (Glatt et al., 2012). Since the basic region in Elp1 was clearly important for Elongator function we hypothesized that it could be involved in binding tRNA molecules to the Elp1–3 subcomplex for wobble uridine modification, given that the Elp3 subunit within this subcomplex is the most likely candidate for carrying out the modification reaction. Elp1 was therefore analysed for potential RNA binding sites using the binding prediction program BindN+, which predicts RNA binding regions from primary sequence based on the properties of RNA binding residues from known structures (Wang and Brown, 2006). This analysis highlighted the conserved carboxy-terminal basic region in both yeast and human Elp1 as being likely to bind RNA (Fig. 6A), consistent with the notion that it may be required for tRNA binding. Such a role might therefore explain the profound consequences of the elp1–KR9A allele on Elongator function.


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)

The Elp1 basic region is predicted to bind RNA and is required for formation of a complex with yeast tRNA.A. Analysis for predicted RNA binding sites within the S. cerevisiae and H. sapiens Elp1 protein sequence using BindN+ (http://bioinfo.ggc.org/bindn+/) showing the high confidence prediction of the basic region as a putative RNA binding region (confidence is scored on a scale of 1–9 with 9 as highest confidence). The locations of the Elp1 basic region and the elp1–KR9A alanine substitution mutations are shown for comparison. Overall conservation between the human and yeast sequences is also indicated (/, identity; •, similar residues).B. Recombinant Elp1 and Elp1–KR9A C-terminal domains (CTDs). Elp1 CTDs with N-terminal GST and C-terminal His6 tags were expressed and purified from E. coli along with GST-His6 as a control. The corresponding proteins were analysed on a 4–12% polyacrylamide gel and stained with instant blue. The GST–Elp1–CTD-His6 fusion proteins are indicated by an arrow.C. Electrophoretic mobility shift assay (EMSA) using ∼ 1 nM 32P-labelled yeast tRNA and the indicated concentrations of recombinant GST, GST–Elp1 and GST–Elp1–KR9A fusion proteins shown in (B). Binding reactions were separated on a 1.5% native agarose gel and analysed by autoradiography. A complex was formed between tRNA and the wild-type Elp1 CTD but not with the Elp1–KR9A CTD.
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fig06: The Elp1 basic region is predicted to bind RNA and is required for formation of a complex with yeast tRNA.A. Analysis for predicted RNA binding sites within the S. cerevisiae and H. sapiens Elp1 protein sequence using BindN+ (http://bioinfo.ggc.org/bindn+/) showing the high confidence prediction of the basic region as a putative RNA binding region (confidence is scored on a scale of 1–9 with 9 as highest confidence). The locations of the Elp1 basic region and the elp1–KR9A alanine substitution mutations are shown for comparison. Overall conservation between the human and yeast sequences is also indicated (/, identity; •, similar residues).B. Recombinant Elp1 and Elp1–KR9A C-terminal domains (CTDs). Elp1 CTDs with N-terminal GST and C-terminal His6 tags were expressed and purified from E. coli along with GST-His6 as a control. The corresponding proteins were analysed on a 4–12% polyacrylamide gel and stained with instant blue. The GST–Elp1–CTD-His6 fusion proteins are indicated by an arrow.C. Electrophoretic mobility shift assay (EMSA) using ∼ 1 nM 32P-labelled yeast tRNA and the indicated concentrations of recombinant GST, GST–Elp1 and GST–Elp1–KR9A fusion proteins shown in (B). Binding reactions were separated on a 1.5% native agarose gel and analysed by autoradiography. A complex was formed between tRNA and the wild-type Elp1 CTD but not with the Elp1–KR9A CTD.
Mentions: The conserved basic region in Elp1 is essential for tRNA wobble uridine modification but despite being capable of functioning as an NLS, it does not appear to influence the distribution of the Elp1 between the nucleus and cytoplasm and is not required for overall assembly of the Elongator complex. In keeping with Elongator's role in tRNA wobble uridine modification, it was recently shown by electrophoretic mobility shift assay (EMSA) that the Elp4–6 hexameric subcomplex of Elongator directly binds tRNAGlu(UUC), consistent with a direct role for Elongator in tRNA binding and subsequent modification (Glatt et al., 2012). Since the basic region in Elp1 was clearly important for Elongator function we hypothesized that it could be involved in binding tRNA molecules to the Elp1–3 subcomplex for wobble uridine modification, given that the Elp3 subunit within this subcomplex is the most likely candidate for carrying out the modification reaction. Elp1 was therefore analysed for potential RNA binding sites using the binding prediction program BindN+, which predicts RNA binding regions from primary sequence based on the properties of RNA binding residues from known structures (Wang and Brown, 2006). This analysis highlighted the conserved carboxy-terminal basic region in both yeast and human Elp1 as being likely to bind RNA (Fig. 6A), consistent with the notion that it may be required for tRNA binding. Such a role might therefore explain the profound consequences of the elp1–KR9A allele on Elongator function.

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