Sequential binding of FurA from Anabaena sp. PCC 7120 to iron boxes: Exploring regulation at the nanoscale María Carmen Pallarés a , Carlos Marcuello a , Laura Botello-Morte b , Andrés González b , María Francisca Fillat b , Anabel Lostao a,c, ⁎ a Laboratorio de Microscopías Avanzadas (LMA), Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, 50018 Zaragoza, Spain b Department of Biochemistry and Molecular and Cell Biology and Institute for Biocomputation and Complex Systems Physics (BiFi), Universidad de Zaragoza, 50009 Zaragoza, Spain c Fundación ARAID, Spain abstract article info Article history: Received 2 October 2013 Received in revised form 6 January 2014 Accepted 8 January 2014 Available online 16 January 2014 Keywords: Ferric uptake regulator Atomic force microscopy Transcriptional regulation Iron box Protein–DNA interaction DNA bending 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 P furA , we examined the topology arrangement of FurA–DNA complexes by atomic force microscopy. Interestingly, FurA–P furA 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 P furA using a sequen- tial mechanism: (i) a monomer specifically binds to an “iron box” and bends P furA ; (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. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Many physiological processes are regulated by DNA-binding proteins that can bind to target sequences on the DNA with high affinity and spec- ificity. Examples of these processes include transcriptional regulation, hormone receptor-mediated activation, and certain types of site-specific recombination. These strong and highly specific interactions use both direct and indirect readout mechanisms and rely on the recognition of the nucleic acid sequence by a protein domain. The mechanism exploits the sequence-dependent local conformation and the mechanical proper- ties of the nucleic acid molecule [1]. Protein-induced DNA deformations, such as looping [2], bending [3,4], and wrapping [5–7], are common occurrence in nucleoprotein complexes and appear to be essential in a variety of gene expression processes and their regulation. An increasing number of prokaryotic and eukaryotic transcription factors have been found to bend the DNA on binding to their specific site. DNA bending can regulate transcription in several ways. It can bring distant bound transcription factors near by means of DNA looping; facilitate the proper orientation of protein factors relative to one another and relative to the promoter, acting as a transcriptional switch [8] and it seems a require- ment for specific site recognition [9]. Since recognition sites in many transcriptional regulators are palindromic sequences, it is assumed that they bind DNA as dimers in order to efficiently regulate their targets [10]. Fur (ferric uptake regulation) proteins are global regulators of prokaryotic metabolism. Among the variety of processes controlled by Fur, the regulation of iron uptake and incorporation was first character- ized in Escherichia coli [11]. It was proposed that Fur worked as a classi- cal repressor using Fe 2+ as co-repressor to bind their DNA targets named “iron boxes” as a dimer. Under iron deficiency, a common situa- tion in many habitats, the release of Fe 2+ from the repressor produces a conformational change in the protein causing its dissociation from DNA and allowing the transcription of genes related to iron metabolism. Fur proteins are ubiquitous among prokaryotes and constitute a superfam- ily whose members are involved in the homeostasis of different metal ions and the defense against oxidative stress. Although they exhibit important functional differences Fur proteins share a common fold in which the C-terminal domain is responsible for dimerization while the N-terminal is involved in recognizing and binding DNA [12]. Usually, Fur proteins are autoregulated and in most cases, the “iron boxes” are Biochimica et Biophysica Acta 1844 (2014) 623–631 Abbreviations: Fur, Ferric uptake regulator; AFM, atomic force microscopy; UV, ultra-violet; SDS, sodium dodecyl-sulfate; DTT, dithiothreitol; EMSA, Electrophoretic mobility shift assays; P furA , furA promoter ⁎ Corresponding author at: Laboratorio de Microscopías Avanzadas (LMA), Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, Ed. I+D+i. Campus Río Ebro. Mariano Esquillor s/n. 50018 Zaragoza, Spain. Tel.: +34 876555357; fax: +34 976762776. E-mail address: aglostao@unizar.es (A. Lostao). 1570-9639/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbapap.2014.01.005 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap