Rapid binding of T7 RNA polymerase is followed by simultaneous bending and opening of the promoter DNA

To form a functional open complex, bacteriophage T7 RNA polymerase (RNAP) binds to its promoter DNA and induces DNA bending and opening. The objective of this study was to elucidate the temporal coupling in DNA binding, bending, and opening processes that occur during initiation. For this purpose, we conducted a combined measurement of stopped-flow fluorescence anisotropy, fluorescence resonance energy transfer (FRET), and 2-aminopurine fluorescence. Stopped-flow anisotropy measurements provided direct evidence of an intermediate resulting from rapid binding of the promoter to T7 RNA polymerase. Stopped-flow FRET measurements showed that promoter bending occurred at a rate constant that was slower than the initial DNA binding rate constant, indicating that the initial complex was not significantly bent. Similarly, stopped-flow 2-aminopurine fluorescence changes showed that promoter opening occurred at a rate constant that was slower than the initial DNA binding rate constant, indicating that the initial complex was not significantly melted. The indistinguishable observed rate constants of FRET and 2-aminopurine fluorescence changes indicate that DNA bending and opening processes are temporally coupled and these DNA conformational changes take place after the DNA binding step. The results in this paper are consistent with the mechanism in which the initial binding of T7 RNAP to the promoter results in a closed complex, which is then converted into an open complex in which the promoter is both sharply bent and melted.     

Microvascular gas embolization clearance following perfluorocarbon administration.

Effective treatment of vascular gas embolism may be possible with emulsified fluorocarbon compounds. We tested the hypothesis that a fluorocarbon emulsion delivered before gas embolization would enhance bubble motion through the vasculature, favoring more rapid clearance. Air microbubbles were injected into the rat cremaster microcirculation in six groups of rats receiving Perftoran, an emulsified fluorocarbon, or saline immediately before, 2 h before, or after bubble injection. Embolism dimensions and dynamics were observed by using intravital microscopy. Surface area at lodging was equal between groups. Bubbles having smaller volume embolized smaller diameter vessels in the Perftoran pretreatment groups. A higher incidence of bubble dislodgement and larger distal displacement occurred in these two groups, with a 36% decrease in the time to bubble clearance and restoration of blood flow. Intravascular emulsified fluorocarbon administration before gas embolization affected initial bubble conformation, increased bubble dislodgement, and resulted in bubble displacement further into the periphery of the microcirculation. These dynamic events did not occur if embolization preceded fluorocarbon administration.