Regulating Gene Expression Through DNA Mechanics: Tightly Looped DNA Represses Transcription.

It is now widely accepted that the mechanical state of DNA can play a major role in regulating the activity of RNA polymerase (RNAP). Not only have the global levels of supercoiling been shown to regulate transcription, but supercoiling has also been implicated in the transcriptional coupling of divergently oriented genes with closely spaced promoters. Additionally, many transcriptional repressors form tight loops of DNA by binding to multiple sites on a DNA template, challenging polymerases to transcribe a DNA template sustaining significant bending curvature. Many studies have provided evidence that the regulatory features of divergent promoter and loop-forming repressor systems share a dependence on the mechanical state of DNA, but these observations have been phenomenological in nature and fail to provide us with a mechanistic understanding of the relationship between RNAP activity as a function of the bending and twisting of DNA in these systems. Consequently, the direct role played by DNA mechanics in these systems remains unclear. I have hypothesized that the mechanical stress within highly bent DNA is itself sufficient to repress transcription. To test this hypothesis, I have developed an assay capable of quantifying the ability of bacteriophage T7 RNAP to transcribe small, circular DNA templates sustaining high levels of bending and torsional stresses. I have characterized both the pre-elongation and elongation kinetics using a highly untwisted 100 bp minicircle, an overtwisted 106 bp minicircle, and a mildly untwisted 108 bp minicircle template. In addition, I have used cryo-electron microscopy to directly observe the topological consequences of the torsional stress sustained within each DNA minicircle species at the single molecule level. Herein, I show that DNA minicircles on the order of 100bp can sustain significant torsional stress without relief by supercoiling, highly bent DNA is directly repressive to transcription, and torsional stress sustained within the DNA template modulates the elongation velocity and processivity of T7 RNAP. The data support a model in which DNA bending can directly control RNAP activity and call for more detailed studies to relate the mechanistic details emerging from this work to regulatory systems known to impart significant bends within the DNA template.Ph.D.Cellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/75859/1/talion_1.pd

Using nanoscale bioreactors to characterize sub-populations of CHO clones and screen transfected pools

Traditional means to quantify growth and production rates for antibody-expressing CHO lines involve sampling aliquots and supernatants from well plates that have been seeded with single cells. The number of clones studied is often limited by cloning efficiencies (typically 5-50%) and the inability to handle large numbers of well plates. The speed at which each clone can be measured is limited by the growth rates of cells and the number of cells required to perform each assay. Both of these factors lead to a practical throughput of 100s of clones screened over the course of 2-4 weeks. Furthermore, each readout of a clone offers little to no insight into the behavior of sub-populations within each clone since the aliquot or supernatant is just a small sample representing the entire population. Please click Additional Files below to see the full abstract

Fabrication of nanoscale zero-mode waveguides using microlithography for single molecule sensing

We present a novel approach to the fabrication of zero-mode waveguides (ZMWs) using inexpensive processing techniques. Our method is capable of rapid fabrication of circular nanoapertures with diameters ranging from 70 nm to 2 μm, allowing us to perform a detailed characterization of the dependence of the fluorescence emission on the waveguide diameter. We also validated the use of the fabricated ZMWs by detecting single molecule binding events with a signal-to-noise ratio of ten.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98608/1/0957-4484_23_45_455301.pd

Cooperative kinking at distant sites in mechanically stressed DNA

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DN

Cooperative kinking at distant sites in mechanically stressed DNA

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA

siRNA-Like Double-Stranded RNAs Are Specifically Protected Against Degradation in Human Cell Extract

RNA interference (RNAi) is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence) any gene of interest by the introduction of synthetic small-interfering (si)RNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs (more precisely, RNA/DNA chimeras) to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET). We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand) double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time