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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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Lemi1 proteins binding to Lemi1 TIRs. Increasing concentrations of the GST–Orf1 protein were incubated with radioactively labelled probes corresponding to the 5′- and 3′-terminal sequences of Lemi1 (Lemi1 TIR1: 106 bp of Lemi1 sequence and 93 bp of flanking genomic DNA; Lemi1 TIR2: 64 bp of Lemi1 sequence and 134 bp of flanking genomic DNA) and were analysed by EMSA. The migrating position of the free probes (F) and the different retarded bands (B1–B3) is shown on both sides of the panel.
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fig4: Lemi1 proteins binding to Lemi1 TIRs. Increasing concentrations of the GST–Orf1 protein were incubated with radioactively labelled probes corresponding to the 5′- and 3′-terminal sequences of Lemi1 (Lemi1 TIR1: 106 bp of Lemi1 sequence and 93 bp of flanking genomic DNA; Lemi1 TIR2: 64 bp of Lemi1 sequence and 134 bp of flanking genomic DNA) and were analysed by EMSA. The migrating position of the free probes (F) and the different retarded bands (B1–B3) is shown on both sides of the panel.

Mentions: Transposase binding to the terminal regions of the mobile element is the first step of the transposition process. In order to test the capacity of the proteins encoded by the original Lemi1 transposon to bind Lemi1 and Emigrant sequences, we reconstructed the consensus Lemi1 coding sequence by replacing the STOP codon found in the Columbia Lemi1 sequence with the tryptophan coding triplet found in Ms-0, RLD, Dijon-D and Tsu-0 ecotypes by site-directed mutagenesis. We expressed in Escherichia coli the two proteins encoded by the modified Lemi1 element as GST fusions: the protein encoded by the first orf (GST–Orf1) and the protein that would be produced by splicing the predicted Lemi1 intron and consisting in a fusion of most of Orf1 and Orf2 (GST–Orf1-2) (Figure 3). On the other hand, we generated a construct containing the insertion found in Coimbra-1 and Coimbra-4 (GST–Orf1-2+) by replacing an AccI–AccI fragment (see Figure 2A) of the Columbia sequence by that of the Coimbra-1. As control proteins, we expressed in E.coli GST fusions with truncated Lemi1 proteins: the GST construct, in which the STOP codon of the Columbia Lemi1 sequence was maintained and that encodes a GST protein fused to a short polypeptide of 38 amino acids, and the GST–BD construct, in which a STOP codon was introduced at position 264 of the protein and that encodes for a GST protein fused with a truncated Orf1 protein that contains the whole DNA-binding domain and a truncated catalytic domain. These proteins were used to perform EMSA with radioactively labelled probes corresponding to the terminal sequences of Lemi1 and Emigrant elements. We first tested the ability of Lemi1 proteins to bind its own terminal sequences. EMSA analysis showed that the GST–Orf1 protein binds Lemi1 TIR1 and TIR2 probes giving one major retarded band, B1 (TIR1), or three retarded bands, B1–B3 (TIR2) (Figure 4), suggesting that the reconstructed protein has retained the ability to specifically bind the transposon TIR sequences. In order to test if Lemi1 proteins could also specifically bind the TIRs of Emigrant elements we performed EMSA analysis with the TIRs of the Emi126 element, a polymorphic Emigrant element belonging to the young EmiA family (11). These analyses showed that Lemi1 proteins specifically bind Emigrant TIRs (Figure 5). Although the control GST protein does not bind to Emi126 probes, the GST–Orf1, GST–Orf1-2 and GST–Orf1-2+ proteins specifically bind to both TIR1 and TIR2 Emi126 probes. Binding to TIR1 gave two retarded bands (B1 and B2, Figure 5A), while binding to TIR2 gave three retarded bands (B1, B2 and B3, Figure 5B). The three proteins tested seem to bind Emigrant probes in a similar way, suggesting that the C-terminal part of the catalytic domain, which coincides with the polypeptide encoded by orf2, does not participate in the binding of Lemi1 protein(s) to DNA. The GST–BD polypeptide binds Emigrant TIR1 and TIR2 sequences but, although its binding of TIR1 is very similar to that of GST–Orf1, GST–Orf1-2 and GST–Orf1-2+ (Figure 5A), its binding to TIR2 appears to be very different. While GST–Orf1, GST–Orf1-2 and GST–Orf1-2+ proteins gave three retarded bands with TIR2, GST–BD gave only one retarded band even at high-protein concentrations (Figure 5B, lanes 5–7). Therefore, although the DNA-binding domain of Lemi1 protein(s) is sufficient for specific binding, a region of the catalytic domain absent from the GST–BD protein is needed for multiple binding to TIR2. A suggestive possibility is that this region, which is absent from the GST–BD polypeptide and is located within the catalytic domain, could mediate protein–protein interactions allowing multiple binding to DNA. Transposase dimerization domains can be located within the DNA-binding domain, e.g. in the mariner-like Sleeping Beauty transposase (15), in the C-terminal part of the protein, in many hAT transposases such as Hermes (16), or within both the DNA-binding domain and the catalytic domain similar to the one in the case of the mariner-like Mos1 transposase (17–19).


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)

Lemi1 proteins binding to Lemi1 TIRs. Increasing concentrations of the GST–Orf1 protein were incubated with radioactively labelled probes corresponding to the 5′- and 3′-terminal sequences of Lemi1 (Lemi1 TIR1: 106 bp of Lemi1 sequence and 93 bp of flanking genomic DNA; Lemi1 TIR2: 64 bp of Lemi1 sequence and 134 bp of flanking genomic DNA) and were analysed by EMSA. The migrating position of the free probes (F) and the different retarded bands (B1–B3) is shown on both sides of the panel.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636448&req=5

fig4: Lemi1 proteins binding to Lemi1 TIRs. Increasing concentrations of the GST–Orf1 protein were incubated with radioactively labelled probes corresponding to the 5′- and 3′-terminal sequences of Lemi1 (Lemi1 TIR1: 106 bp of Lemi1 sequence and 93 bp of flanking genomic DNA; Lemi1 TIR2: 64 bp of Lemi1 sequence and 134 bp of flanking genomic DNA) and were analysed by EMSA. The migrating position of the free probes (F) and the different retarded bands (B1–B3) is shown on both sides of the panel.
Mentions: Transposase binding to the terminal regions of the mobile element is the first step of the transposition process. In order to test the capacity of the proteins encoded by the original Lemi1 transposon to bind Lemi1 and Emigrant sequences, we reconstructed the consensus Lemi1 coding sequence by replacing the STOP codon found in the Columbia Lemi1 sequence with the tryptophan coding triplet found in Ms-0, RLD, Dijon-D and Tsu-0 ecotypes by site-directed mutagenesis. We expressed in Escherichia coli the two proteins encoded by the modified Lemi1 element as GST fusions: the protein encoded by the first orf (GST–Orf1) and the protein that would be produced by splicing the predicted Lemi1 intron and consisting in a fusion of most of Orf1 and Orf2 (GST–Orf1-2) (Figure 3). On the other hand, we generated a construct containing the insertion found in Coimbra-1 and Coimbra-4 (GST–Orf1-2+) by replacing an AccI–AccI fragment (see Figure 2A) of the Columbia sequence by that of the Coimbra-1. As control proteins, we expressed in E.coli GST fusions with truncated Lemi1 proteins: the GST construct, in which the STOP codon of the Columbia Lemi1 sequence was maintained and that encodes a GST protein fused to a short polypeptide of 38 amino acids, and the GST–BD construct, in which a STOP codon was introduced at position 264 of the protein and that encodes for a GST protein fused with a truncated Orf1 protein that contains the whole DNA-binding domain and a truncated catalytic domain. These proteins were used to perform EMSA with radioactively labelled probes corresponding to the terminal sequences of Lemi1 and Emigrant elements. We first tested the ability of Lemi1 proteins to bind its own terminal sequences. EMSA analysis showed that the GST–Orf1 protein binds Lemi1 TIR1 and TIR2 probes giving one major retarded band, B1 (TIR1), or three retarded bands, B1–B3 (TIR2) (Figure 4), suggesting that the reconstructed protein has retained the ability to specifically bind the transposon TIR sequences. In order to test if Lemi1 proteins could also specifically bind the TIRs of Emigrant elements we performed EMSA analysis with the TIRs of the Emi126 element, a polymorphic Emigrant element belonging to the young EmiA family (11). These analyses showed that Lemi1 proteins specifically bind Emigrant TIRs (Figure 5). Although the control GST protein does not bind to Emi126 probes, the GST–Orf1, GST–Orf1-2 and GST–Orf1-2+ proteins specifically bind to both TIR1 and TIR2 Emi126 probes. Binding to TIR1 gave two retarded bands (B1 and B2, Figure 5A), while binding to TIR2 gave three retarded bands (B1, B2 and B3, Figure 5B). The three proteins tested seem to bind Emigrant probes in a similar way, suggesting that the C-terminal part of the catalytic domain, which coincides with the polypeptide encoded by orf2, does not participate in the binding of Lemi1 protein(s) to DNA. The GST–BD polypeptide binds Emigrant TIR1 and TIR2 sequences but, although its binding of TIR1 is very similar to that of GST–Orf1, GST–Orf1-2 and GST–Orf1-2+ (Figure 5A), its binding to TIR2 appears to be very different. While GST–Orf1, GST–Orf1-2 and GST–Orf1-2+ proteins gave three retarded bands with TIR2, GST–BD gave only one retarded band even at high-protein concentrations (Figure 5B, lanes 5–7). Therefore, although the DNA-binding domain of Lemi1 protein(s) is sufficient for specific binding, a region of the catalytic domain absent from the GST–BD protein is needed for multiple binding to TIR2. A suggestive possibility is that this region, which is absent from the GST–BD polypeptide and is located within the catalytic domain, could mediate protein–protein interactions allowing multiple binding to DNA. Transposase dimerization domains can be located within the DNA-binding domain, e.g. in the mariner-like Sleeping Beauty transposase (15), in the C-terminal part of the protein, in many hAT transposases such as Hermes (16), or within both the DNA-binding domain and the catalytic domain similar to the one in the case of the mariner-like Mos1 transposase (17–19).

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