Immobilization of biotinylated DNA on 2-D streptavidin crystals

The structural study of transient nucleoprotein complexes by electron microscopy is hampered by the coexistence of multiple interaction states leading to an heterogeneous image population. To tackle this problem, we have investigated the controlled immobilization of double stranded DNA molecules and of nucleoprotein complexes onto a support suitable for cryo-electron microscopy observation. The DNA was end-labeled with a biotin moiety in order to decorate, or to be incorporated into, two-dimensional streptavidin crystals formed in contact of a biotinylated lipid layer. The binding specificity and efficiency were examined by radioactively labeled oligonucleotides and by direct visualization of unstained and hydrated nucleic acid molecules in cryo-electron microscopy. By using RNA polymerase we further show that, once immobilized, femtomolar amounts of DNA template are suitable to interact with the enzyme. The image analysis of the RNA polymerase-DNA complexes showed that a three-dimensional model can be retrieved from such samples

Structure of SAGA and mechanism of TBP deposition on gene promoters

International audienceSAGA (Spt–Ada–Gcn5–acetyltransferase) is a 19-subunit complex that stimulates transcription via two chromatin-modifying enzymatic modules and by delivering the TATA box binding protein (TBP) to nucleate the pre-initiation complex on DNA, a pivotal event in the expression of protein-encoding genes1. Here we present the structure of yeast SAGA with bound TBP. The core of the complex is resolved at 3.5 Å resolution (0.143 Fourier shell correlation). The structure reveals the intricate network of interactions that coordinate the different functional domains of SAGA and resolves an octamer of histone-fold domains at the core of SAGA. This deformed octamer deviates considerably from the symmetrical analogue in the nucleosome and is precisely tuned to establish a peripheral site for TBP, where steric hindrance represses binding of spurious DNA. Complementary biochemical analysis points to a mechanism for TBP delivery and release from SAGA that requires transcription factor IIA and whose efficiency correlates with the affinity of DNA to TBP. We provide the foundations for understanding the specific delivery of TBP to gene promoters and the multiple roles of SAGA in regulating gene expressio

Atomic structure of the SAGA complex and it's interaction with TBP

The transcription of eukaryotic protein genes is controlled by a plethora of proteins which act together in multi-component complexes to facilitate the DNA dependent RNA polymerase II (Pol II) enzyme to bind to the transcription start site and to generate messenger RNA from the gene's coding sequence. The protein that guides the transcription machinery to the exact transcription start site is called TATA-box Binding Protein, or TBP. TBP is part of two large protein complexes involved in Pol II transcription, TFIID and SAGA. The two complexes share several subunits implicated in the interaction with TBP and contain proteins with structural elements highly homologous to nucleosomal histones. Despite the intensive study of transcription initiation, the mode of interaction of TBP with these complexes and its release upon DNA binding was elusive. In this study we demonstrate the quasi-atomic model of SAGA in complex with TBP. The structure reveals the intricate network of interactions that coordinate the different functional domains of SAGA and resolves a deformed octamer of histone-fold domains at the core of SAGA. This deformed octamer is precisely tuned to establish a peripheral site for TBP binding, where it is protected by steric hindrance against the binding of spurious DNA. Complementary biochemical analysis points to a mechanism for TBP delivery and release from SAGA that requires the general transcription factor TFIIA and whose efficiency correlates with the affinity of DNA to TBP.As the TBP binding machinery is highly similar in TFIID and SAGA, we demonstrated a universal mechanism of how TBP is delivered to gene promoters during transcription initiation

Filamentous condensation of DNA induced by pegylated poly-L-lysine and transfection efficiency.

International audienceIn this paper we propose a detailed analysis of structural and morphological properties of two poly-L-lysine (PLL)-based transfection formulations, PLL/DNA and pegylated PLL (PLL-g-PEG)/DNA, by means of atomic force microscopy (AFM) and transmission electron microscopy (TEM). Comparing PLL-g-PEG/DNA with PLL/DNA polyplexes, we demonstrate that, due to the presence of PEG, the particles differ not only in size, shape, and crystalline structure, but also in transfection efficiency. While PLL condensates DNA in large agglomerates, PLL grafted with polyethylene glycol 2000 can condensate DNA in long filaments with diameters of some nanometers (6-20 nm). These structures are dependent on the grafting ratio and are more efficient than compacted ones, showing that DNA uptake and processing by cell is directly related to physicochemical properties of the polyplexes