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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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The elp1–KR9A mutation does not disrupt Elongator complex assembly but causes subtle differences in association with Elp5 and Kti12.A. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains expressing HA-tagged Elp3, Elp5 or Kti12. All samples were analysed with anti–GFP and anti-HA. Input samples were also analysed using anti-Cdc28 as a loading control. Arg4–GFP was confirmed to have similar pulldown efficiency to Elp1–GFP and was used as a control to demonstrate that interaction between Elp1 and the various HA tagged complex members was specific. All pulldown samples were analysed from the same blot. It should be noted that Elp1 is always observed by Western blot analysis as both a full-length and a faster migrating form (truncated at its N-terminus) that may be a degradation product or serve an as yet unknown function (Fichtner et al., 2003).B. Quantification of co-immunoprecipitation efficiency. Immunoprecipitation of HA-tagged proteins was quantified by densitometry of the HA tag signals and normalized using the Elp1–GFP signals across the indicated number of replicates (n), setting the value for the wild-type strain in each case to 1.0. Error bars represent the standard deviation of the mean and the significance of the differences was analysed using a one way ANOVA, with ‘*’ indicating a P-value of < 0.05 (significant) and ‘***’ indicating a P-value of < 0.001 (very highly significant). Any small differences in Elp3 association were not statistically significant when analysed in this manner.
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fig04: The elp1–KR9A mutation does not disrupt Elongator complex assembly but causes subtle differences in association with Elp5 and Kti12.A. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains expressing HA-tagged Elp3, Elp5 or Kti12. All samples were analysed with anti–GFP and anti-HA. Input samples were also analysed using anti-Cdc28 as a loading control. Arg4–GFP was confirmed to have similar pulldown efficiency to Elp1–GFP and was used as a control to demonstrate that interaction between Elp1 and the various HA tagged complex members was specific. All pulldown samples were analysed from the same blot. It should be noted that Elp1 is always observed by Western blot analysis as both a full-length and a faster migrating form (truncated at its N-terminus) that may be a degradation product or serve an as yet unknown function (Fichtner et al., 2003).B. Quantification of co-immunoprecipitation efficiency. Immunoprecipitation of HA-tagged proteins was quantified by densitometry of the HA tag signals and normalized using the Elp1–GFP signals across the indicated number of replicates (n), setting the value for the wild-type strain in each case to 1.0. Error bars represent the standard deviation of the mean and the significance of the differences was analysed using a one way ANOVA, with ‘*’ indicating a P-value of < 0.05 (significant) and ‘***’ indicating a P-value of < 0.001 (very highly significant). Any small differences in Elp3 association were not statistically significant when analysed in this manner.

Mentions: GFP–Trap immunoprecipitations of Elp1–GFP and Elp1–KR9A–GFP were assayed for association with HA tagged Kti12, Elp3 and Elp5 and these were each present as co-precipitants under physiological salt conditions (Fig. 4A). This confirmed both that the Elp1–3 sub complex was intact and that interactions with Elp5 and Kti12 are not abolished by the basic region mutation, and so the Elp1 basic region is not required for the overall gross assembly of the Elongator complex. However, when these interactions were analysed carefully by densitometry, Elp1–KR9A–GFP was consistently found associated with ∼ 30% less Elp5-3HA and ∼ 40% less Kti12-3HA than the wild-type Elp1–GFP protein (Fig. 4B). These minor interaction defects are highly consistent and statistically significant. In contrast, Elp3 levels and the interaction between Elp1–KR9A–GFP and Elp3-3HA appeared largely unchanged (Fig. 4B), consistent with earlier work in which Elp3 was found to become very unstable in strains where it could not interact with Elp1 (Petrakis et al., 2004). The small defects in Kti12 and Elp5 interactions with the Elp1–KR9A mutant may reflect the fact that Elongator is no longer functional. For example, if the complex needs to shuttle between multiple forms and is stalled due to the basic region mutations, this may lead to reduced complex stability and subsequently affect the interaction with other subunits.


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–KR9A mutation does not disrupt Elongator complex assembly but causes subtle differences in association with Elp5 and Kti12.A. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains expressing HA-tagged Elp3, Elp5 or Kti12. All samples were analysed with anti–GFP and anti-HA. Input samples were also analysed using anti-Cdc28 as a loading control. Arg4–GFP was confirmed to have similar pulldown efficiency to Elp1–GFP and was used as a control to demonstrate that interaction between Elp1 and the various HA tagged complex members was specific. All pulldown samples were analysed from the same blot. It should be noted that Elp1 is always observed by Western blot analysis as both a full-length and a faster migrating form (truncated at its N-terminus) that may be a degradation product or serve an as yet unknown function (Fichtner et al., 2003).B. Quantification of co-immunoprecipitation efficiency. Immunoprecipitation of HA-tagged proteins was quantified by densitometry of the HA tag signals and normalized using the Elp1–GFP signals across the indicated number of replicates (n), setting the value for the wild-type strain in each case to 1.0. Error bars represent the standard deviation of the mean and the significance of the differences was analysed using a one way ANOVA, with ‘*’ indicating a P-value of < 0.05 (significant) and ‘***’ indicating a P-value of < 0.001 (very highly significant). Any small differences in Elp3 association were not statistically significant when analysed in this manner.
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fig04: The elp1–KR9A mutation does not disrupt Elongator complex assembly but causes subtle differences in association with Elp5 and Kti12.A. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains expressing HA-tagged Elp3, Elp5 or Kti12. All samples were analysed with anti–GFP and anti-HA. Input samples were also analysed using anti-Cdc28 as a loading control. Arg4–GFP was confirmed to have similar pulldown efficiency to Elp1–GFP and was used as a control to demonstrate that interaction between Elp1 and the various HA tagged complex members was specific. All pulldown samples were analysed from the same blot. It should be noted that Elp1 is always observed by Western blot analysis as both a full-length and a faster migrating form (truncated at its N-terminus) that may be a degradation product or serve an as yet unknown function (Fichtner et al., 2003).B. Quantification of co-immunoprecipitation efficiency. Immunoprecipitation of HA-tagged proteins was quantified by densitometry of the HA tag signals and normalized using the Elp1–GFP signals across the indicated number of replicates (n), setting the value for the wild-type strain in each case to 1.0. Error bars represent the standard deviation of the mean and the significance of the differences was analysed using a one way ANOVA, with ‘*’ indicating a P-value of < 0.05 (significant) and ‘***’ indicating a P-value of < 0.001 (very highly significant). Any small differences in Elp3 association were not statistically significant when analysed in this manner.
Mentions: GFP–Trap immunoprecipitations of Elp1–GFP and Elp1–KR9A–GFP were assayed for association with HA tagged Kti12, Elp3 and Elp5 and these were each present as co-precipitants under physiological salt conditions (Fig. 4A). This confirmed both that the Elp1–3 sub complex was intact and that interactions with Elp5 and Kti12 are not abolished by the basic region mutation, and so the Elp1 basic region is not required for the overall gross assembly of the Elongator complex. However, when these interactions were analysed carefully by densitometry, Elp1–KR9A–GFP was consistently found associated with ∼ 30% less Elp5-3HA and ∼ 40% less Kti12-3HA than the wild-type Elp1–GFP protein (Fig. 4B). These minor interaction defects are highly consistent and statistically significant. In contrast, Elp3 levels and the interaction between Elp1–KR9A–GFP and Elp3-3HA appeared largely unchanged (Fig. 4B), consistent with earlier work in which Elp3 was found to become very unstable in strains where it could not interact with Elp1 (Petrakis et al., 2004). The small defects in Kti12 and Elp5 interactions with the Elp1–KR9A mutant may reflect the fact that Elongator is no longer functional. For example, if the complex needs to shuttle between multiple forms and is stalled due to the basic region mutations, this may lead to reduced complex stability and subsequently affect the interaction with other subunits.

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