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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

Binding of the Elp1 C-terminal domain to tRNA occurs with a dissociation constant in the low micromolar range and is not competed by poly(U) or ssDNA.A. Electrophoretic mobility shift assay (EMSA) using ∼ 1 nM 32P-labelled yeast tRNA, 0.4 μM recombinant Elp1 C-terminal domain and the indicated molar excess of unlabelled yeast tRNA, poly(U) or ssDNA.B. EMSA using ∼ 0.1 nM 32P-labelled yeast tRNA and the indicated concentrations of recombinant Elp1 C-terminal domain. Bound and free tRNA bands were quantified from three replicate experiments and used to estimate a dissociation constant (KD) of 0.55 μM for the interaction by non-linear curve fitting (f = a*xb/[cb + xb]). The Hill coefficient (b) for the fitted curve was 1.76 ± 0.19. All binding reactions were separated on a 1.5% native agarose gel and analysed by autoradiography.
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fig07: Binding of the Elp1 C-terminal domain to tRNA occurs with a dissociation constant in the low micromolar range and is not competed by poly(U) or ssDNA.A. Electrophoretic mobility shift assay (EMSA) using ∼ 1 nM 32P-labelled yeast tRNA, 0.4 μM recombinant Elp1 C-terminal domain and the indicated molar excess of unlabelled yeast tRNA, poly(U) or ssDNA.B. EMSA using ∼ 0.1 nM 32P-labelled yeast tRNA and the indicated concentrations of recombinant Elp1 C-terminal domain. Bound and free tRNA bands were quantified from three replicate experiments and used to estimate a dissociation constant (KD) of 0.55 μM for the interaction by non-linear curve fitting (f = a*xb/[cb + xb]). The Hill coefficient (b) for the fitted curve was 1.76 ± 0.19. All binding reactions were separated on a 1.5% native agarose gel and analysed by autoradiography.

Mentions: To test the specificity of the interaction between the Elp1–CTD and tRNA, competition binding experiments were carried out (Fig. 7A). While addition of unlabelled yeast tRNA at a 100-fold excess efficiently competed formation of the [32P]-tRNA–Elp1–CTD complex, addition of a similar excess of either poly(U) or ssDNA left complex formation between the tRNA probe and Elp1–CTD essentially unaffected. The Elp1 C-terminal domain therefore preferentially binds tRNA over poly(U) RNA or ssDNA. We also compared the ability of tRNA purified from either wild-type or elp1Δ mutant yeast (in which wobble uridine-containing tRNAs lack the Elongator-dependent mcm5 and ncm5 modifications) as competitors of [32P]-tRNA binding to the Elp1 CTD. However, no differences were found (data not shown), suggesting that there are unlikely to be very large differences in affinity resulting from whether or not the tRNA carries Elongator-dependent modifications.


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)

Binding of the Elp1 C-terminal domain to tRNA occurs with a dissociation constant in the low micromolar range and is not competed by poly(U) or ssDNA.A. Electrophoretic mobility shift assay (EMSA) using ∼ 1 nM 32P-labelled yeast tRNA, 0.4 μM recombinant Elp1 C-terminal domain and the indicated molar excess of unlabelled yeast tRNA, poly(U) or ssDNA.B. EMSA using ∼ 0.1 nM 32P-labelled yeast tRNA and the indicated concentrations of recombinant Elp1 C-terminal domain. Bound and free tRNA bands were quantified from three replicate experiments and used to estimate a dissociation constant (KD) of 0.55 μM for the interaction by non-linear curve fitting (f = a*xb/[cb + xb]). The Hill coefficient (b) for the fitted curve was 1.76 ± 0.19. All binding reactions were separated on a 1.5% native agarose gel and analysed by autoradiography.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150532&req=5

fig07: Binding of the Elp1 C-terminal domain to tRNA occurs with a dissociation constant in the low micromolar range and is not competed by poly(U) or ssDNA.A. Electrophoretic mobility shift assay (EMSA) using ∼ 1 nM 32P-labelled yeast tRNA, 0.4 μM recombinant Elp1 C-terminal domain and the indicated molar excess of unlabelled yeast tRNA, poly(U) or ssDNA.B. EMSA using ∼ 0.1 nM 32P-labelled yeast tRNA and the indicated concentrations of recombinant Elp1 C-terminal domain. Bound and free tRNA bands were quantified from three replicate experiments and used to estimate a dissociation constant (KD) of 0.55 μM for the interaction by non-linear curve fitting (f = a*xb/[cb + xb]). The Hill coefficient (b) for the fitted curve was 1.76 ± 0.19. All binding reactions were separated on a 1.5% native agarose gel and analysed by autoradiography.
Mentions: To test the specificity of the interaction between the Elp1–CTD and tRNA, competition binding experiments were carried out (Fig. 7A). While addition of unlabelled yeast tRNA at a 100-fold excess efficiently competed formation of the [32P]-tRNA–Elp1–CTD complex, addition of a similar excess of either poly(U) or ssDNA left complex formation between the tRNA probe and Elp1–CTD essentially unaffected. The Elp1 C-terminal domain therefore preferentially binds tRNA over poly(U) RNA or ssDNA. We also compared the ability of tRNA purified from either wild-type or elp1Δ mutant yeast (in which wobble uridine-containing tRNAs lack the Elongator-dependent mcm5 and ncm5 modifications) as competitors of [32P]-tRNA binding to the Elp1 CTD. However, no differences were found (data not shown), suggesting that there are unlikely to be very large differences in affinity resulting from whether or not the tRNA carries Elongator-dependent modifications.

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