Limits...
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.

Show MeSH

Related in: MedlinePlus

Self-association of Elp1 is unaffected by the elp1–KR9A mutation and is independent of the assembly of the Elp4–6 hexamer.A. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains containing empty vector (EV), YCplac111–ELP1–6HA or YCplac111–elp1–KR9A–6HA expressing HA-tagged wild-type or mutant Elp1 respectively. All samples were analysed by Western blotting with anti–GFP and anti-HA. Inputs were also analysed using anti-Cdc28 as a loading control.B. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains containing empty vector (EV), YCplac111–ELP1–6HA or YCplac111–elp1–KR9A–6HA expression plasmids either with or without deletion of the ELP5 gene (elp5Δ).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150532&req=5

fig05: Self-association of Elp1 is unaffected by the elp1–KR9A mutation and is independent of the assembly of the Elp4–6 hexamer.A. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains containing empty vector (EV), YCplac111–ELP1–6HA or YCplac111–elp1–KR9A–6HA expressing HA-tagged wild-type or mutant Elp1 respectively. All samples were analysed by Western blotting with anti–GFP and anti-HA. Inputs were also analysed using anti-Cdc28 as a loading control.B. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains containing empty vector (EV), YCplac111–ELP1–6HA or YCplac111–elp1–KR9A–6HA expression plasmids either with or without deletion of the ELP5 gene (elp5Δ).

Mentions: Since it was recently shown that Elongator forms a dodecamer containing two copies each of its six subunits (Glatt et al., 2012), it was possible that Elp1–KR9A could be defective in assembly into this higher order complex. To test this, YCp–ELP1–6HA and YCp–elp1–KR9A–6HA expression plasmids were transformed into ELP1–GFP and elp1–KR9A–GFP strains to generate cells in which two differentially tagged copies of Elp1 were present. The ELP1–GFP strain remained sensitive to zymocin following introduction of YCp–ELP1–6HA, confirming that the increased ELP1 copy number did not cause significant loss of function (data not shown). Wild-type Elp1–GFP and Elp1–KR9A–GFP were immunoprecipitated using GFP–Trap and assayed for co-precipitation of the 6HA-tagged versions of Elp1 (Fig. 5A). GFP-tagged wild-type Elp1 co-precipitated Elp1–6HA as previously reported (Glatt et al., 2012) and could also associate to a similar extent with Elp1–KR9A–6HA. In the reciprocal experiment, GFP tagged Elp1–KR9A also co-precipitated both Elp1–6HA and Elp1–KR9A–6HA, again with no obvious defect in the interaction between any of the different combinations. Thus there is no apparent defect in Elp1 self-association within the Elongator complex due to the Elp1 basic region mutation.


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)

Self-association of Elp1 is unaffected by the elp1–KR9A mutation and is independent of the assembly of the Elp4–6 hexamer.A. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains containing empty vector (EV), YCplac111–ELP1–6HA or YCplac111–elp1–KR9A–6HA expressing HA-tagged wild-type or mutant Elp1 respectively. All samples were analysed by Western blotting with anti–GFP and anti-HA. Inputs were also analysed using anti-Cdc28 as a loading control.B. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains containing empty vector (EV), YCplac111–ELP1–6HA or YCplac111–elp1–KR9A–6HA expression plasmids either with or without deletion of the ELP5 gene (elp5Δ).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: Self-association of Elp1 is unaffected by the elp1–KR9A mutation and is independent of the assembly of the Elp4–6 hexamer.A. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains containing empty vector (EV), YCplac111–ELP1–6HA or YCplac111–elp1–KR9A–6HA expressing HA-tagged wild-type or mutant Elp1 respectively. All samples were analysed by Western blotting with anti–GFP and anti-HA. Inputs were also analysed using anti-Cdc28 as a loading control.B. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains containing empty vector (EV), YCplac111–ELP1–6HA or YCplac111–elp1–KR9A–6HA expression plasmids either with or without deletion of the ELP5 gene (elp5Δ).
Mentions: Since it was recently shown that Elongator forms a dodecamer containing two copies each of its six subunits (Glatt et al., 2012), it was possible that Elp1–KR9A could be defective in assembly into this higher order complex. To test this, YCp–ELP1–6HA and YCp–elp1–KR9A–6HA expression plasmids were transformed into ELP1–GFP and elp1–KR9A–GFP strains to generate cells in which two differentially tagged copies of Elp1 were present. The ELP1–GFP strain remained sensitive to zymocin following introduction of YCp–ELP1–6HA, confirming that the increased ELP1 copy number did not cause significant loss of function (data not shown). Wild-type Elp1–GFP and Elp1–KR9A–GFP were immunoprecipitated using GFP–Trap and assayed for co-precipitation of the 6HA-tagged versions of Elp1 (Fig. 5A). GFP-tagged wild-type Elp1 co-precipitated Elp1–6HA as previously reported (Glatt et al., 2012) and could also associate to a similar extent with Elp1–KR9A–6HA. In the reciprocal experiment, GFP tagged Elp1–KR9A also co-precipitated both Elp1–6HA and Elp1–KR9A–6HA, again with no obvious defect in the interaction between any of the different combinations. Thus there is no apparent defect in Elp1 self-association within the Elongator complex due to the Elp1 basic region mutation.

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