Interaction of the plant homeodomain protein ATH1 with DNA.

IVANA VIOLA; RAQUEL CHAN; DANIEL GONZALEZ

Iguazú. Misiones

Congreso; XL Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2004

SAIB

Abstract ATH1 is a light-regulated Arabidopsis gene that encodes a member of the Bell family of plant TALE homeodomain (HD) proteins. The HD is a conserved compact domain that directly contacts DNA. In this study, the ATH1 binding sequence, TGACAGGT, was determined by selecting binding sites from random-sequence oligonucleotides through several rounds of binding and amplification (SELEX). In vitro DNA binding assays with oligonucleotides containing variations of the consensus sequence were performed with ATH1 and STM (a class I Knox HD protein). These assays indicated that both HDs recognize similar sequences with different affinity and that changes within the GAC core deeply affect their interaction with DNA. A more detailed picture of the binding of these proteins to its target site was obtained by footprinting and missing nucleoside experiments using hydroxyl radical cleavage of DNA. Exchange of residues present at position 54 of the HDs of ATH1 and STM, Val and Lys, respectively, indicated that this residue is a determinant of binding affinity. Changes at positions 5 and 6 of the TGACAGGT sequence significantly affect the interaction when position 54 is Val, while position 7 is very important for all proteins. The results indicate that ATH1 and STM bind similar sequences with different affinity and that subtle differences exist in their interaction with DNA. Residue 54 would be in part responsible for the different properties of both HDs.