Molecular architecture of fur binding to iron-induced and - repressed genes in Helicobacter pylori

The ferric uptake regulator protein Fur regulates iron-dependent gene expression in bacteria. In the human pathogen Helicobacter pylori, Fur has been shown to regulate iron-induced and iron-repressed genes. Herein we investigate the molecular mechanisms that control this differential iron-responsive Fur regulation. Hydroxyl radical footprinting showed that Fur has different binding architectures, which characterize distinct operator typologies. On operators recognized with higher affinity by holo-Fur, the protein binds to a continuous AT-rich stretch of about 20 bp, displaying an extended protection pattern. This is indicative of protein wrapping around the DNA helix. DNA binding interference assays with the minor groove binding drug distamycin A, point out that the recognition of the holo-operators occurs through the minor groove of the DNA. By contrast, on the apo-operators, Fur binds primarily to thymine dimers within a newly identified TCATTn10TT consensus element, indicative of Fur binding to one side of the DNA, in the major groove of the double helix. Reconstitution of the TCATTn10TT motif within a holo-operator results in a feature binding swap from an holo-Fur- to an apo-Fur-recognized operator, affecting both affinity and binding architecture of Fur, and conferring apo-Fur repression features in vivo. Size exclusion chromatography indicated that Fur is a dimer in solution. However, in the presence of divalent metal ions the protein is able to multimerize. Accordingly, apo-Fur binds DNA as a dimer in gel shift assays, while in presence of iron, higher order complexes are formed. Stoichiometric Ferguson analysis indicates that these complexes correspond to one or two Fur tetramers, each bound to an operator element. Together these data suggest that the apo- and holo-Fur repression mechanisms apparently rely on two distinctive modes of operator-recognition, involving respectively the readout of a specific nucleotide consensus motif in the major groove for apo-operators, and the recognition of AT-rich stretches in the minor groove for holo-operators, whereas the iron-responsive binding affinity is controlled through metal-dependent shaping of the protein structure in order to match preferentially the major or the minor groove.

La via Emilia tra Rimini e Forlì. Ruolo e immagine della strada

Abstract Il tema delle infrastrutture, intese come parte dell’architettura dello spazio urbano e del territorio, assume un ruolo centrale in molti progetti contemporanei e costituisce la ragione di questa ricerca. E’ preso in esame, in particolare, il tracciato extraurbano della via Emilia, antica strada consolare romana la cui definizione risale al II sec. a.C., nel tratto compreso tra le città di Rimini e Forlì. Studiare la strada nel suo rapporto con il territorio locale ha significato in primo luogo prendere in considerazione la via Emilia in quanto manufatto, ma anche in quanto percorso che si compie nel tempo. Si è dunque cercato di mostrare come, in parallelo all’evoluzione della sua sezione e della geometria del suo tracciato, sia cambiata anche la sua fruizione, e come si sia evoluto il modo in cui la strada viene “misurata”, denominata e gestita. All’interno di una riflessione critica sulla forma e sul ruolo della strada nel corso dei secoli la Tesi rilegge il territorio nella sua dimensione di “palinsesto”, riconoscendo e isolando alcuni momenti in cui la via Emilia ha assunto un valore “simbolico” che rimanda alla Roma imperiale. La perdita del significato via Emilia, intesa come elemento di “costruzione” del territorio, ha origine con il processo di urbanizzazione diffusa che ha investito il territorio extraurbano a partire dalla fine della seconda guerra mondiale. La condizione attuale della strada, sempre più congestionata dal traffico veicolare, costituisce la premesse per una riflessione sul futuro della sua forma e degli insediamenti che attraversa. La strategia proposta dagli Enti locali che prevede il raddoppio della strada, con la costruzione della via Emilia Bis, non garantisce solo un potenziamento infrastrutturale ma rappresenta l’occasione per sottrarre al tracciato attuale la funzione di principale asse di comunicazione extraurbana. La via Emilia potrebbe così recuperare il ruolo di itinerario narrativo, attraverso la configurazione dei suoi spazi collettivi, l’architettura dei suoi edifici, il significato dei suoi monumenti, e diventare spazio privilegiato di relazione e di aggregazione. The theme of urban infrastructures, thought as part of the design of urban space and territory, has a central role in several contemporary projects and is the reason of this research. The object of the study is the extra urban route of the via Emilia, an ancient roman road which has been defined in the II century b. C., in its stretch between the cities of Rimini and Forlì. Studying the road in its relationship with the local environment has meant first of all considering the via Emilia as an “artefact” but also as a path that takes place over time. The aim of this research was also to demonstrate how its fruition has changed together with the evolution of the section and geometry of the route, and how the road itself is measured, named and managed. Within a critical approach on the shape and on the role played by the road through the centuries, this Essay reinterprets the territory in its dimension of “palimpsest”, identifying and isolating some periods of time when the via Emilia assumed a symbolic value which recalls the Imperial Rome. The loss of the meaning of the via Emilia, intended as an element that “constitutes” the territory originates from a process of diffused urbanization, which spread in the extra urban environment from the end of the second world war. The actual condition of the road, more and more congested by traffic, is the premise of a reflection about the future of its shape and of the settlements alongside. The strategy proposed by the local authorities, that foresees to double the size of the road, building the via Emilia Bis, not only guarantees an infrastructural enhancement but also it represents an opportunity to take off from the road itself the current function of being the principal axis of extra urban connection. In this way the via Emilia could regain its role as a narrative itinerary, through the configuration of its public spaces, the architecture of its buildings, the meaning of its monuments, and then become a privileged space of relationship and aggregation.