Biochemical characterization and DNA binding properties of the MocR family member GabR, a PLP-dependent transcription factor

GabR è un fattore di trascrizione chimerico appartenente alla famiglia dei MocR/GabR, costituito da un dominio N-terminale elica-giro-elica di legame al DNA e un dominio effettore e/o di oligomerizzazione al C-terminale. I due domini sono connessi da un linker flessibile di 29 aminoacidi. Il dominio C-terminale è strutturalmente omologo agli enzimi aminotransferasici fold-type I, i quali, utilizzando il piridossal-5’-fosfato (PLP) come cofattore, sono direttamente coinvolti nel metabolismo degli aminoacidi. L’interazione contemporanea di PLP e acido γ-aminobutirrico (GABA) a GabR fa sì che questa promuova la trascrizione di due geni, gabT e gabD, implicati nel metabolismo del GABA. GabR cristallizza come un omodimero con una configurazione testa-coda. Il legame con la regione promotrice gabTD avviene attraverso il riconoscimento specifico di due sequenze dirette e ripetute (ATACCA), separate da uno spacer di 34 bp. In questo studio sono state indagate le proprietà biochimiche, strutturali e di legame al DNA della proteina GabR di Bacillus subtilis. L’analisi spettroscopica dimostra che GabR interagisce con il PLP formando l’aldimina interna, mentre in presenza di GABA si ottiene l’aldimina esterna. L’interazione fra il promotore gabTD e le forme holo e apo di GabR è stata monitorata mediante Microscopia a Forza atomica (AFM). In queste due condizioni di legame è stata stimata una Kd di circa 40 ηM. La presenza di GABA invece, determinava un incremento di circa due volte della Kd, variazioni strutturali nei complessi GabR-DNA e una riduzione del compattamento del DNA alla proteina, indipendentemente dalla sequenza del promotore in esame. Al fine di valutare il ruolo delle caratteristiche topologiche del promotore, sono state inserite cinque e dieci bp all’interno della regione spacer che separa le due sequenze ripetute dirette riconosciute da GabR. I significativi cambiamenti topologici riscontrati nel frammento aggiunto di cinque bp si riflettono anche sulla forte riduzione dell’affinità di legame verso la proteina. Al contrario, l’inserzione di 10 bp provoca solamente l’allontanamento delle sequenze ripetute dirette. L’assenza quindi di cambiamenti significativi nella topologia di questo promotore fa sì che l’affinità di legame per GabR rimanga pressoché inalterata rispetto al promotore non mutato. L’analisi del potenziale elettrostatico superficiale di GabR mostra la presenza di una fascia carica positivamente che si estende lungo un’intera faccia della proteina. Per verificare l’importanza di questa caratteristica di GabR nel meccanismo di interazione al DNA, sono stati preparati ed indagati i mutanti R129Q e K362-366Q, in cui la carica positiva superficiale risultava indebolita. L’affinità di legame dei mutanti di GabR per il DNA era inferiore rispetto alla proteina non mutata, in particolar modo nel mutante K362-366Q. Le evidenze acquisite suggeriscono che la curvatura intrinseca del promotore ed il corretto orientamento delle sequenze sulla doppia elica, più della distanza che le separa, siano critici per sostenere l’interazione con GabR. Oltre a questo, la superficie positiva di GabR è richiesta per accomodare la curvatura del DNA sul corpo della proteina. Alla luce di questo, l’interazione GabR-gabTD è un esempio di come il riconoscimento specifico di sequenze, la topologia del DNA e le caratteristiche strutturali della proteina siano contemporaneamente necessarie per sostenere un’interazione proteina-DNA stabile.

The tectonically-confined Firenzuola turbidite system (Miocene, Marnoso-arenacea Formation, northern Apennines, Italy): an example of facies variations related to the basin morphology

The Firenzuola turbidite system formed during a paroxysmal phase of thrust propagation, involving the upper Serravallian deposits of the Marnoso-arenacea Formation (MAF). During this phase the coeval growth of two major tectonic structures, the M. Castellaccio thrust and the Verghereto high, played a key role, causing a closure of the inner basin and a coeval shift of the depocentre to the outer basin. This work focuses on this phase of fragmentation of the MAF basin; it is based on a new detailed high-resolution stratigraphic framework, which was used to determine the timing of growth of the involved structures and their direct influence on sediment dispersal and on the lateral and vertical turbidite facies distribution. The Firenzuola turbidite system stratigraphy is characterized by the occurrence of mass-transport complexes (MTCs) and thick sandstone accumulation in the depocentral area, which passes to finer drape over the structural highs; the differentiation between these two zones increases over time and ends with the deposition of marly units over the structural highs and the emplacement of the Visignano MTC. According to the stratigraphic pattern and turbidite facies characteristics, the Firenzuola System has been split into two phases, namely Firenzuola I and Firenzuola II: the former is quite similar to the underlying deposits, while the latter shows the main fragmentation phase, testifying the progressive isolation of the inner basin and a coeval shift of the depocentre to the outer basin. The final stratigraphic and sedimentological dataset has been used to create a quantitative high-resolution 3D facies distribution using the Petrel software platform. This model allows a detailed analysis of lateral and vertical facies variations that can be exported to several reservoirs settings in hydrocarbon exploration and exploitation areas, since facies distributions and geometries of the reservoir bodies of many sub-surface turbidite basins show a significant relationship to the syndepositional structural activity, but are beyond seismic resolution.

Stratigrafia fisica ed analisi di facies dei depositi torbiditici della Formazione delle Arenarie del Monte Cervarola tra la Val Secchia e la Val Scoltenna (Aquitaniano-Burdigaliano, Appennino Settentrionale)

The Cervarola Sandstones Formation (CSF), Aquitanian-Burdigalian in age, was deposited in an elongate, NW-stretched foredeep basin formed in front of the growing Northern Apennines orogenic wedge. The stratigraphic succession of the CSF, in the same way of other Apennine foredeep deposits, records the progressive closure of the basin due to the propagation of thrust fronts toward north-east, i.e. toward the outer and shallower foreland ramp. This process produce a complex foredeep characterized by synsedimentary structural highs and depocenters that can strongly influence the lateral and vertical turbidite facies distribution. Of consequence the main aim of this work is to describe and discuss this influence on the basis of a new high-resolution stratigraphic framework performed by measuring ten stratigraphic logs, for a total thickness of about 2000m, between the Secchia and Scoltenna Valleys (30km apart). In particular, the relationship between the turbidite sedimentation and the ongoing tectonic activity during the foredeep evolution has been describe through various stratigraphic cross sections oriented parallel and perpendicular to the main tectonic structures. On the basis of the high resolution physical stratigraphy of the studied succession, we propose a facies tract and an evolutionary model for the Cervarola Sandstones in the studied area. Thanks to these results and the analogies with others foredeep deposits of the northern Apennines, such as the Marnoso-arenacea Formation, the Cervarola basin has been interpreted as a highly confined foredeep controlled by an intense synsedimentary tectonic activity. The most important evidences supporting this hypothesis are: 1) the upward increase, in the studied stratigraphic succession (about 1000m thick), of sandstone/mudstone ratio, grain sizes and Ophiomorpha-type trace fossils testifying the high degree of flow deceleration related to the progressive closure and uplift of the foredeep. 2) the occurrence in the upper part of the stratigraphic succession of coarse-grained massive sandstones overlain by tractive structures such as megaripples and traction carpets passing downcurrent into fine-grained laminated contained-reflected beds. This facies tract is interpreted as related to deceleration and decoupling of bipartite flows with the deposition of the basal dense flows and bypass of the upper turbulent flows. 3) the widespread occurrence of contained reflected beds related to morphological obstacles created by tectonic structures parallel and perpendicular to the basin axis (see for example the Pievepelago line). 4) occurrence of intra-formational slumps, constituted by highly deformed portion of fine-grained succession, indicating a syn-sedimentary tectonic activity of the tectonic structures able to destabilize the margins of the basin. These types of deposits increase towards the upper part of the stratigraphic succession (see points 1 and 2) 5) the impressive lateral facies changes between intrabasinal topographic highs characterized by fine-grained and thin sandstone beds and marlstones and depocenters characterized by thick to very thick coarse-grained massive sandstones. 6) the common occurrence of amalgamation surfaces, flow impact structures and mud-draped scours related to sudden deceleration of the turbidite flows induced by the structurally-controlled confinement and morphological irregularities. In conclusion, the CSF has many analogies with the facies associations occurring in other tectonically-controlled foredeeps such as those of Marnoso-arenacea Formation (northern Italy) and Annot Sandstones (southern France) showing how thrust fronts and transversal structures moving towards the foreland, were able to produce a segmented foredeep that can strongly influence the turbidity current deposition.