Sequential binding of FurA from Anabaena sp. PCC 7120 to iron boxes: exploring regulation at the nanoscale.
Fur (ferric uptake regulator) proteins are involved in the control of a variety of processes in most prokaryotes. Although it is assumed that this regulator binds its DNA targets as a dimer, the way in which this interaction occurs remains unknown. We have focused on FurA from the cyanobacterium Anabaena sp. PCC 7120. To assess the molecular mechanism by which FurA specifically binds to "iron boxes" in PfurA, we examined the topology arrangement of FurA-DNA complexes by atomic force microscopy. Interestingly, FurA-PfurA complexes exhibit several populations, in which one is the predominant and depends clearly on the regulator/promoter ratio on the environment. Those results together with EMSA and other techniques suggest that FurA binds PfurA using a sequential mechanism: (i) a monomer specifically binds to an "iron box" and bends PfurA; (ii) two situations may occur, that a second FurA monomer covers the free "iron box" or that joins to the previously used forming a dimer which would maintain the DNA kinked; (iii) trimerization in which the DNA is unbent; and (iv) finally undergoes a tetramerization; the next coming molecules cover the DNA strands unspecifically. In summary, the bending appears when an "iron box" is bound to one or two molecules and decreases when both "iron boxes" are covered. These results suggest that DNA bending contributes at the first steps of FurA repression promoting the recruitment of new molecules resulting in a fine regulation in the Fur-dependent cluster associated genes.
Laboratorio de Microscopías Avanzadas (LMA), Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, 50018 Zaragoza, Spain.
María Carmen
MC
Pallarés
Laboratorio de Microscopías Avanzadas (LMA), Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, 50018 Zaragoza, Spain.
Carlos
C
Marcuello
Department of Biochemistry and Molecular and Cell Biology and Institute for Biocomputation and Complex Systems Physics (BiFi), Universidad de Zaragoza, 50009 Zaragoza, Spain.
Laura
L
Botello-Morte
Department of Biochemistry and Molecular and Cell Biology and Institute for Biocomputation and Complex Systems Physics (BiFi), Universidad de Zaragoza, 50009 Zaragoza, Spain.
Andrés
A
González
Department of Biochemistry and Molecular and Cell Biology and Institute for Biocomputation and Complex Systems Physics (BiFi), Universidad de Zaragoza, 50009 Zaragoza, Spain.
María Francisca
MF
Fillat
Laboratorio de Microscopías Avanzadas (LMA), Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, 50018 Zaragoza, Spain; Fundación ARAID, Spain. Electronic address: aglostao@unizar.es.
Anabel
A
Lostao
D017033:Anabaena / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D007501:Iron / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D036103:Nanotechnology; D011485:Protein Binding
D001426:Bacterial Proteins; D007501:Iron
Atomic force microscopy; DNA bending; Ferric uptake regulator; Iron box; Protein–DNA interaction; Transcriptional regulation