Bianca Sclavi

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We have used time-resolved x-ray-generated hydroxyl radical footprinting to directly characterize, at single-nucleotide resolution, several intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase on the T7A1 promoter at 37 degrees C. Three sets of intermediates, corresponding to two major conformational changes, are(More)
The formation of a transcriptionally active complex by RNA polymerase involves a series of short-lived structural intermediates where protein conformational changes are coupled to DNA wrapping and melting. We have used time-resolved KMnO(4) and hydroxyl-radical X-ray footprinting to directly probe conformational signatures of these complexes at the T7A1(More)
Radiolysis of water with a synchrotron x-ray beam permits the hydroxyl radical-accessible surface of an RNA to be mapped with nucleotide resolution in 10 milliseconds. Application of this method to folding of the Tetrahymena ribozyme revealed that the most stable domain of the tertiary structure, P4-P6, formed cooperatively within 3 seconds. Exterior(More)
We designed a microfluidic chemostat consisting of 600 sub-micron trapping/growth channels connected to two feeding channels. The microchemostat traps E. coli cells and forces them to grow in lines for over 50 generations. Excess cells, including the mother cells captured at the start of the process, are removed from both ends of the growth channels by the(More)
Hydroxyl radicals (.OH) can cleave the phosphodiester backbone of nucleic acids and are valuable reagents in the study of nucleic acid structure and protein-nucleic acid interactions. Irradiation of solutions by high flux "white light" X-ray beams based on bending magnet beamlines at the National Synchrotron Light Source (NSLS) yields sufficient(More)
In most bacteria, the timing and synchrony of initiation of chromosomal replication are determined by the binding of the AAA(+) protein DnaA to a set of high- and low-affinity sites found within the origin of chromosomal replication (oriC). Despite the large amount of information on the role and regulation of DnaA, the actual structure of the DnaA-oriC(More)
DNA methyltransferases of the Dam family (including bacteriophage T4-encoded Dam DNA (adenine-N(6))-methyltransferase (T4Dam)) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. In this study, we describe the application of direct(More)
In Escherichia coli, overlapping rounds of DNA replication allow the bacteria to double in faster times than the time required to copy the genome. The precise timing of initiation of DNA replication is determined by a regulatory circuit that depends on the binding of a critical number of ATP-bound DnaA proteins at the origin of replication, resulting in the(More)
All organisms that synthesize their own DNA have evolved mechanisms for maintaining a constant DNA/cell mass ratio independent of growth rate. The DNA/cell mass ratio is a central parameter in the processes controlling the cell cycle. The co-ordination of DNA replication with cell growth involves multiple levels of regulation. DNA synthesis is initiated at(More)