Transcription initiation by RNA polymerase (RNP) carrying the house-keeping σ subunit, σ⁷⁰ (Eσ⁷⁰), is repressed by H-NS at a number of promoters including hdeABp in Escherichia coli, while initiation with RNP carrying the stationary phase σ, σ³⁸ (Eσ³⁸), is not. We investigated the molecular mechanism of selective repression by H-NS to identify the differences in transcription initiation by the two forms of RNPs, which show indistinguishable promoter selectivities in vitro. Using hdeABp as a model promoter, we observed with purified components that H-NS, acting at a sequence centered at -118, selectively repressed transcription by Eσ⁷⁰. This selective repression is attributed to the differences in the interactions between hdeABp and the two forms of RNPs, since no other factor is required for the repression. We observed that the two forms of RNPs could form an open initiation complex (RP_O) at hdeABp, but that Eσ⁷⁰ failed to initiate transcription in the presence of H-NS. Interestingly, KMnO₄ assays and high-resolution atomic force microscopy (AFM) revealed that hdeABp DNA wrapped around Eσ⁷⁰ more tightly than around Eσ³⁸, resulting in the potential crossing over of the DNA arms that project out of Eσ⁷⁰ · RP_O but not out of Eσ³⁸ · RP_O. Based on these observations, we postulated that H-NS bound at -118 laterally extends by the cooperative recruitment of H-NS molecules to the promoter-downstream sequence joined by wrapping of the DNA around Eσ⁷⁰ · RP_O, resulting in effective sealing of the DNA loop and trapping of Eσ⁷⁰. Such a ternary complex of H-NS · Eσ⁷⁰hdeABp was demonstrated by AFM. In this case, therefore, Eσ⁷⁰ acts as a cofactor for DNA looping. Expression of this class of genes by Eσ³⁸ in the stationary phase is not due to its promoter specificity but to the architecture of the promoter · Eσ³⁸ complex.

Diabetic retinopathy is predominantly caused by vascular endothelial growth factor (VEGF)–induced vascular leakage; however, the underlying mechanism is unclear. Here we designed an in vivo transglutaminase (TGase) activity assay in mouse retina and demonstrated that hyperglycemia induced vascular leakage by activating TGase2 in diabetic retina. VEGF elevated TGase2 activity through sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) concentrations in endothelial cells. The TGase inhibitors cystamine and monodansylcadaverin or TGase2 small interfering RNA (siRNA) prevented VEGF-induced stress fiber formation and vascular endothelial (VE)–cadherin disruption, which play a critical role in modulating endothelial permeability. Intravitreal injection of two TGase inhibitors or TGase2 siRNA successfully inhibited hyperglycemia-induced TGase activation and microvascular leakage in the retinas of diabetic mice. C-peptide or ROS scavengers also inhibited TGase activation in diabetic mouse retinas. The role of TGase2 in VEGF-induced vascular leakage was further supported using diabetic TGase2−/− mice. Thus, our findings suggest that ROS-mediated activation of TGase2 plays a key role in VEGF-induced vascular leakage by stimulating stress fiber formation and VE-cadherin disruption.