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Binding of the Vestigial co-factor switches the DNA-target selectivity of the Scalloped selector protein

Georg Halder*,{ddagger} and Sean B. Carroll{ddagger}

Howard Hughes Medical Institute and Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison,WI 53706, USA
* Present address: The University of Texas, MD Anderson Cancer Center, Biochemistry and Molecular Biology, 1515 Holcombe Boulevard, Box 117, Houston, TX 77030-4095, USA



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  		Fig. 1. Vg binding switches the DNA-target specificity of Sd. EMSAs using in vitro transcribed and translated (TNT) Sd and Vg proteins binding to templates shown in Table 1. Four lanes of EMSA are shown for each DNA template. From left to right, DNA templates are incubated with unprogrammed TNT extract (lanes 1,5,9,13,17); Sd alone (lanes 2,6,10,14,18); Sd co-translated with Vg (lanes 3,7,11,15,19); and Vg alone (lanes 4,8,12,16,20). Unprogrammed TNT extract does not shift any of these probes. Sd alone binds poorly to doublet sites in cut-564 (lane 2), kni-268 (lane 6) and sal-762 (lane 10), but Vg-Sd complexes binds well to all three probes (lanes 3,7, 11); Sd alone binds to single sites in sal-862 (lane 14) and 1xGT, but co-expression of Sd with Vg did not result in higher order complexes (lanes 15,19). On the contrary, Vg inhibited Sd from binding and reduced the amount of Sd-DNA complexes observed. The residual binding activity migrates at the position of the Sd-DNA complexes and is thus due to uncomplexed Sd. None of the probes are bound by Vg in the absence of Sd. F, free probe. Proteins expressed and probes used are indicated above gels.

 


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  		Fig. 2. Cooperativity of Sd binding is not required for Vg-Sd complex formation on DNA. (A) EMSAs of Sd, Sd and Vg, and Vg binding to the 2xGT and GTspaceGT probes. Sd bound to the 2xGT template as a monomer and as a dimer (Sd and Sd2; lane 2). Incubation of a co-translated mixture of Sd and Vg produced an additional complex that migrated more slowly (VgnSd2; lane 3), while expression of Vg alone did not result in any detectable DNA-binding activity (lane 4). The Vg-Sd complex bound to GTspaceGT with similar affinity as to 2xGT (lanes 3,7), although cooperativity of Sd binding is reduced in the GTspaceGT probe, as it does not bind two molecules of Sd, in contrast to 2xGT (lanes 2,6). Labeling and arrangement of lanes is as in Fig. 1. (B) EMSAs showing titrations of purified TEA domain binding to 2xGT and GTspaceGT. Both probes are shifted by 1 ng TEA domain added and thus have similar affinities. However, two TEA molecules bind cooperatively to 2xGT but non-cooperatively to GTspaceGT. TEA, one molecule TEA domain bound; TEA2, two TEA molecules bound. Protein concentrations are indicated in ng/20 µl. F, free probe.

 


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  		Fig. 3. Domains conserved between fly and vertebrate Vg homologs have different functions in Sd binding and Vg-Sd complex formation on target DNA. (A) Schematic structure of Drosophila Vg and its homologs from Mosquito (Mos-Vg) and human: Fondue (h-Fdu) and Tondu (h-Tdu), respectively. Conserved domains are boxed in color: the Sd interaction domain (SID) in red: and the N- and C-terminal domains in green and orange, respectively. Protein lengths are to scale and an amino acid ruler is shown at the bottom. (B) Sequence comparison of the conserved domains between Dros-Vg, Mos-Vg, h-Fdu and h-Tdu. Identical residues are boxed in color and indicated by a dot. Percent identity over entire domains are indicated to the right. Dros-Vg and h-Fdu have an intron at similar positions in the SID (arrowheads). Residues shared between h-Fdu and h-Tdu are boxed in blue. (C) Schematic of the series of Vg deletion mutants tested for interaction with Sd in solution and for Vg-Sd complex formation on DNA. A summary of their activities is indicated on the right.

 


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  		Fig. 4. Vg protein domains outside the Sd interaction domain are required for Vg-Sd complex formation on DNA but not for Vg-Sd interaction in solution. (A) EMSA of the binding to the cut template by the Vg deletion mutant proteins co-expressed with Sd. Only the two internal deletions {Delta}(73-176) and {Delta}(73-274) are as efficient in DNA complex formation as full-length Vg (lanes 3,10,11). The deletions 66-C, SID-C and N-SID showed partial activity, while all other deletions were not active. Lane 1, TNT extract; lane 2, Sd only; lanes 3-13 Sd co-expressed with the indicated Vg mutants. Labeling as in Fig. 1. (B) SDS-PAGE of Co-IPs of Sd and Vg mutants. The 35S-labeled proteins produced by TNT (T) and the precipitated proteins (IP) are shown next to each other for each Vg mutant. Lanes 1 and 2: anti-Myc antibody does not precipitate untagged Vg or Sd. Lanes 3-24: precipitation of Myc-tagged Sd with anti-Myc antibody co-precipitates all Vg mutants, except the mutant deleted for SID (lanes 5,6). Top band is Sd, lower bands are Vg mutant proteins.

 


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  		Fig. 5. Vg and Sd form multimeric complexes on DNA but heterodimers in solution. (A) Lanes 1-3: EMSA with VgHA and/or Vg{Delta}(73-274) co-expressed with Sd binding to the cut template. Black arrowhead indicates complexes with intermediate mobility, presumably comprising the two Vg forms, Sd and DNA. Open arrowhead indicates complexes with the same mobility as those observed by co-expressing full-length Vg and Sd only. Lanes 4-6: same reactions incubated with anti-HA antibody. Complexes containing full-length Vg (lanes 4,5) as well as the intermediate complexes (lane 5, arrowhead), but not the untagged Vg{Delta}(73-274) (lane 6), are supershifted. Vg{Delta}(73-176) gave the same effect (not shown). (B) SDS-PAGE of Co-IPs of Sd co-expressed with VgHA and/or Vg{Delta}(73-176). Precipitation of VgHA co-precipitated Sd but not the other Vg form demonstrating that Vg and Sd form complexes in solution that do not contain more than one Vg molecule. The anti-HA antibody did not precipitate the untagged Vg deletion (lanes 5,6). The Vg{Delta}(73-274) protein gave the same result (not shown).

 


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  		Fig. 6. Vg-Sd complex formation on DNA does not require bases outside of the Sd-binding sites. (A) Top strands of the series of truncated templates. TT and AA bases at the ends of the probes were added in order to label the probes with 32P {alpha}-dATP. The extent of the Sd binding sites (shaded regions) was defined by systematic mutational analysis (Butler and Ordahl, 1999). (B) EMSA using TNT produced Vg and Sd and the probes shown in A. The 31 and 25 mers bind strongly to Sd and the Vg-Sd complex (lanes 1-7). The 21 mer is weakly bound by Sd (lane 8), while the Vg-Sd complex forms nearly as efficiently as on the longer templates. The 18 mer is not bound by Sd or Vg-Sd. Labeling is as in Fig. 1.

 


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  		Fig. 7. Model for Vg-Sd interaction and DNA-binding selectivity. (A) Sd binds to A- but not B-sites in cells that express Sd. (B) Sd forms a 1:1 complex with Vg in developing wing cells that express both proteins. The interaction of Sd with Vg prevents Sd from binding to A-sites. However, the Vg-Sd complex is able to bind to B-sites. This activity requires two B-sites in close proximity. Binding to B-sites may be accompanied by conformational changes in Vg-Sd that are only induced when two B-sites are present, indicating that interactions take place between neighboring Vg-Sd complexes.

 

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