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The proteins encoded by the pogo-like Lemi1 element bind the TIRs and subterminal repeated motifs of the Arabidopsis Emigrant MITE: consequences for the transposition mechanism of MITEs.

Loot C, Santiago N, Sanz A, Casacuberta JM - Nucleic Acids Res. (2006)

Bottom Line: We present here evidence for a recent mobility of the Arabidopsis Emigrant MITE and we report on the capacity of the proteins encoded by the related Lemi1 transposon, a pogo-related element, to specifically bind Emigrant elements.Our results show that Lemi1 proteins bind Emigrant TIRs but also bind cooperatively to subterminal repeated motifs.The requirement of internal sequences for the formation of proper DNA/protein structure could affect the capacity of divergent MITEs to be mobilized by distantly related transposases.

View Article: PubMed Central - PubMed

Affiliation: Departament de Genètica Molecular, Laboratori de Genètica Molecular Vegetal, CSIC-IRTA, Jordi Girona 18, 08034 Barcelona, Spain.

ABSTRACT
MITEs (miniature inverted-repeated transposable elements) are a particular class of defective DNA transposons usually present within genomes as high copy number populations of highly homogeneous elements. Although an active MITE, the mPing element, has recently been characterized in rice, the transposition mechanism of MITEs remains unknown. It has been proposed that transposases of related transposons could mobilize MITEs in trans. Moreover, it has also been proposed that the presence of conserved terminal inverted-repeated (TIR) sequences could be the only requirement of MITEs for mobilization, allowing divergent or unrelated elements to be mobilized by a particular transposase. We present here evidence for a recent mobility of the Arabidopsis Emigrant MITE and we report on the capacity of the proteins encoded by the related Lemi1 transposon, a pogo-related element, to specifically bind Emigrant elements. This suggests that Lemi1 could mobilize Emigrant elements and makes the Lemi1/Emigrant couple an ideal system to study the transposition mechanism of MITEs. Our results show that Lemi1 proteins bind Emigrant TIRs but also bind cooperatively to subterminal repeated motifs. The requirement of internal sequences for the formation of proper DNA/protein structure could affect the capacity of divergent MITEs to be mobilized by distantly related transposases.

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Footprinting analysis of the Lemi1 proteins binding to Emi126. Increasing concentrations of the GST–Orf1-2 were incubated with radioactively labelled Emi126 TIR1 (A) and TIR2 (B) probes. The position of the TIR sequence is shown by a grey arrow and the position of the TIR and subterminal repeated motifs are indicated by solid arrows. The position of G nucleotides of the upper strand (TIR2) or the bottom strand (corresponding to C nucleotides in the upper strand) (TIR1), revealed by DMS reactions, are shown on the right of each gel. G (or C) positions, numbered from the first nucleotide of the TIR, as in Figure 5, are 1, 9, 10, 12, 34, 37 and 80 (TIR1), and −23, −22, −13, −12, −6, 1, 9, 10, 12, 25, 27, 41, 42, 44, 61 and 64 (TIR2). A closed triangle indicates the DNase I hypersensible position flanking the protected region in TIR2. A sequence comparison of the TIRs and the repeated motifs is shown at the bottom. Identical nucleotides appear as white letters on black boxes.
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fig7: Footprinting analysis of the Lemi1 proteins binding to Emi126. Increasing concentrations of the GST–Orf1-2 were incubated with radioactively labelled Emi126 TIR1 (A) and TIR2 (B) probes. The position of the TIR sequence is shown by a grey arrow and the position of the TIR and subterminal repeated motifs are indicated by solid arrows. The position of G nucleotides of the upper strand (TIR2) or the bottom strand (corresponding to C nucleotides in the upper strand) (TIR1), revealed by DMS reactions, are shown on the right of each gel. G (or C) positions, numbered from the first nucleotide of the TIR, as in Figure 5, are 1, 9, 10, 12, 34, 37 and 80 (TIR1), and −23, −22, −13, −12, −6, 1, 9, 10, 12, 25, 27, 41, 42, 44, 61 and 64 (TIR2). A closed triangle indicates the DNase I hypersensible position flanking the protected region in TIR2. A sequence comparison of the TIRs and the repeated motifs is shown at the bottom. Identical nucleotides appear as white letters on black boxes.

Mentions: We performed DNase I footprinting analysis to determine the Lemi1-binding sites in Emi126. These experiments showed that GST–Orf1-2 binds Emigrant TIRs but also other internal sequences (Figure 7). In the case of TIR2 the DNase I protection covers a continuous region of 56 bp including the TIR and two repeated motifs that coincide with the 3′-half of the TIR sequence (Figure 7B and see Figure 5B for sequence details). The TIR2 footprint is flanked by a DNase I hypersensitive band indicating that binding of Lemi1 protein(s) induce(s) a distortion of the target DNA. Protein binding often affects DNA structure and in particular transposases often distort DNA upon binding (22). The DNase I footprinting analysis of TIR1 shows a protection that covers two regions: 23 bp of the TIR itself and a 29 bp region consisting of two repeats of a sequence coinciding with the 3′-half of the TIR in opposite orientation and separated from the TIR by 22 bp (Figure 7A and see Figure 5A for sequence details).


The proteins encoded by the pogo-like Lemi1 element bind the TIRs and subterminal repeated motifs of the Arabidopsis Emigrant MITE: consequences for the transposition mechanism of MITEs.

Loot C, Santiago N, Sanz A, Casacuberta JM - Nucleic Acids Res. (2006)

Footprinting analysis of the Lemi1 proteins binding to Emi126. Increasing concentrations of the GST–Orf1-2 were incubated with radioactively labelled Emi126 TIR1 (A) and TIR2 (B) probes. The position of the TIR sequence is shown by a grey arrow and the position of the TIR and subterminal repeated motifs are indicated by solid arrows. The position of G nucleotides of the upper strand (TIR2) or the bottom strand (corresponding to C nucleotides in the upper strand) (TIR1), revealed by DMS reactions, are shown on the right of each gel. G (or C) positions, numbered from the first nucleotide of the TIR, as in Figure 5, are 1, 9, 10, 12, 34, 37 and 80 (TIR1), and −23, −22, −13, −12, −6, 1, 9, 10, 12, 25, 27, 41, 42, 44, 61 and 64 (TIR2). A closed triangle indicates the DNase I hypersensible position flanking the protected region in TIR2. A sequence comparison of the TIRs and the repeated motifs is shown at the bottom. Identical nucleotides appear as white letters on black boxes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636448&req=5

fig7: Footprinting analysis of the Lemi1 proteins binding to Emi126. Increasing concentrations of the GST–Orf1-2 were incubated with radioactively labelled Emi126 TIR1 (A) and TIR2 (B) probes. The position of the TIR sequence is shown by a grey arrow and the position of the TIR and subterminal repeated motifs are indicated by solid arrows. The position of G nucleotides of the upper strand (TIR2) or the bottom strand (corresponding to C nucleotides in the upper strand) (TIR1), revealed by DMS reactions, are shown on the right of each gel. G (or C) positions, numbered from the first nucleotide of the TIR, as in Figure 5, are 1, 9, 10, 12, 34, 37 and 80 (TIR1), and −23, −22, −13, −12, −6, 1, 9, 10, 12, 25, 27, 41, 42, 44, 61 and 64 (TIR2). A closed triangle indicates the DNase I hypersensible position flanking the protected region in TIR2. A sequence comparison of the TIRs and the repeated motifs is shown at the bottom. Identical nucleotides appear as white letters on black boxes.
Mentions: We performed DNase I footprinting analysis to determine the Lemi1-binding sites in Emi126. These experiments showed that GST–Orf1-2 binds Emigrant TIRs but also other internal sequences (Figure 7). In the case of TIR2 the DNase I protection covers a continuous region of 56 bp including the TIR and two repeated motifs that coincide with the 3′-half of the TIR sequence (Figure 7B and see Figure 5B for sequence details). The TIR2 footprint is flanked by a DNase I hypersensitive band indicating that binding of Lemi1 protein(s) induce(s) a distortion of the target DNA. Protein binding often affects DNA structure and in particular transposases often distort DNA upon binding (22). The DNase I footprinting analysis of TIR1 shows a protection that covers two regions: 23 bp of the TIR itself and a 29 bp region consisting of two repeats of a sequence coinciding with the 3′-half of the TIR in opposite orientation and separated from the TIR by 22 bp (Figure 7A and see Figure 5A for sequence details).

Bottom Line: We present here evidence for a recent mobility of the Arabidopsis Emigrant MITE and we report on the capacity of the proteins encoded by the related Lemi1 transposon, a pogo-related element, to specifically bind Emigrant elements.Our results show that Lemi1 proteins bind Emigrant TIRs but also bind cooperatively to subterminal repeated motifs.The requirement of internal sequences for the formation of proper DNA/protein structure could affect the capacity of divergent MITEs to be mobilized by distantly related transposases.

View Article: PubMed Central - PubMed

Affiliation: Departament de Genètica Molecular, Laboratori de Genètica Molecular Vegetal, CSIC-IRTA, Jordi Girona 18, 08034 Barcelona, Spain.

ABSTRACT
MITEs (miniature inverted-repeated transposable elements) are a particular class of defective DNA transposons usually present within genomes as high copy number populations of highly homogeneous elements. Although an active MITE, the mPing element, has recently been characterized in rice, the transposition mechanism of MITEs remains unknown. It has been proposed that transposases of related transposons could mobilize MITEs in trans. Moreover, it has also been proposed that the presence of conserved terminal inverted-repeated (TIR) sequences could be the only requirement of MITEs for mobilization, allowing divergent or unrelated elements to be mobilized by a particular transposase. We present here evidence for a recent mobility of the Arabidopsis Emigrant MITE and we report on the capacity of the proteins encoded by the related Lemi1 transposon, a pogo-related element, to specifically bind Emigrant elements. This suggests that Lemi1 could mobilize Emigrant elements and makes the Lemi1/Emigrant couple an ideal system to study the transposition mechanism of MITEs. Our results show that Lemi1 proteins bind Emigrant TIRs but also bind cooperatively to subterminal repeated motifs. The requirement of internal sequences for the formation of proper DNA/protein structure could affect the capacity of divergent MITEs to be mobilized by distantly related transposases.

Show MeSH
Related in: MedlinePlus