INVESTIGADORES
VIOLA Ivana Lorena
congresos y reuniones científicas
Título:
DNA binding properties of a Knotted transcription factor from sunflower.
Autor/es:
MARIANA F. TIONI; MA. LAURA CABEZA; VIOLA IVANA; DANIEL H. GONZALEZ; RAQUEL L. CHAN
Lugar:
Villa Carlos Paz, Córdoba. Argentina
Reunión:
Congreso; XXXVIII Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2002
Institución organizadora:
SAIB
Resumen:
The Knotted family was the first group of homeodomain (HD) containing transcription factors identified in plants. The HD is a 60-aminoacid conserved structure that contains three ƒ¿-helices and is responsible for DNA binding. In our laboratory, we are studying the properties of HAKN1, a HD containing protein that belongs to the knotted family of sunflower, with special insight in the identification of the amino acids that contact specific bases in the DNA. Using PCR-mediated mutagenesis we obtained a series of mutants with point changes in different positions of the HD. We expressed the wild type and mutant proteins as fusions with the maltose binding protein of Escherichia coli. The purified proteins were used in EMSAs with five different DNA probes that contained either the preferred sequence core bound by this class of proteins (5L-TGTGAC-3L) or point variations of it or its surrounding bases. Our results show that position 55 of the HD is crucial for proper DNA binding as the mutant R55A was unable to bind any of the tested probes. Besides, the gGh in the fourth position of the DNA core is essential for recognition by HAKN1 since the probe with a single change in this position was not bound by any of the proteins. Finally, the wild type HD showed a marked preference for one of the probes while the mutant I50S bound a different probe more strongly, suggesting that position 50 in the HD participates in the recognition of specific bases in the DNA.