The structural basis of DNA binding specificity within a transcription factor family

Auxin is a phytohormone important for different growth and developmental processes in plants. Auxin’s signaling pathway has been for long studied and its main proteins identified. The final effectors of this signaling pathway are transcription factors that will regulate the transcription of different target genes. These transcription factors are members of a large family: ARFs. This family is conserved in all plant species and in Arabidopsis consists of 23 members. Some of these ARFs work repressing their target genes and others activating them. Some of them are restricted to certain tissues while others are expressed all over the plant, generating specific subsets of ARFs in different plant tissues (Fig. 1) (Rademacher et al, 2011). Moreover they can be involved in completely different processes, and mutations in some of them show severe phenotypes, like in the case of the well-studied ARF5/MP; plants with a defect only in this ARF won’t be able to make a root (Fig. 2) (Berleth and Jürgens, 1993).

Figure 1. Expression pattern of some ARFs in Arabidopsis embryos. Rademacher et al, 2011
Figure 1. Expression pattern of some ARFs in Arabidopsis embryos. Rademacher et al, 2011
Figure 2. An arf5 mutant plant is unable to make a root.
Figure 2. An arf5 mutant plant is unable to make a root.

How each of these transcription factors regulate specific target genes is not known and what confers this specificity is one of the main questions of this project.             By means of a structural biology approach we are looking at a molecular level how ARFs interact with DNA and other proteins; identifying key aminoacids for their function. The properties of selected ARFs are tested by biochemical/in vitro techniques as well as in living plants. By these means we can observe the effect of mutating selected aminoacids.

To find optimal DNA recognition sites we have chosen a microarray technique that allows us to test the transcription factors against every possible 6mer occurring in the genome (Fig. 3) (Godoy et al, 2011). Since not many direct target genes are known for ARFs we would like to identify them by the use of Chip-Seq, in this way we could also screen for recognition sequences with the help of bioinformatics.

Figure 3. Protein Binding Microarray (PBM) will give us the binding sequence prefered by our transcription factors.
Figure 3. Protein Binding Microarray (PBM) will give us the binding sequence prefered by our transcription factors.

To further understand how the recognition of target genes happens, we are also interested in identifying other proteins that may interact with ARFs forming complexes that would regulate transcription.

Techniques: protein isolation, site directed mutagenesis, FRET-FLIM, Protein Binding Microarray, Surface Plasmon Resonance, Chip-seq, IP-MS.