Transcription by RNA polymerase II (Pol II) is a complex process that requires general transcription factors and Pol II to assemble on DNA into preinitiation complexes that can begin RNA synthesis upon binding of NTPs (nucleoside triphosphate). The pathways by which preinitiation complexes form, and how this impacts transcriptional activity are not completely clear. To address these issues, we developed a single molecule system using TIRF (total internal reflection fluorescence) microscopy and purified human transcription factors, which allows us to visualize transcriptional activity at individual template molecules. We see that stable interactions between polymerase II (Pol II) and a heteroduplex DNA template do not depend on general transcription factors; however, transcriptional activity is highly dependent upon TATA-binding protein, TFIIB and TFIIF. We also found that subsets of general transcription factors and Pol II can form stable complexes that are precursors for functional transcription complexes upon addition of the remaining factors and DNA. Ultimately we found that Pol II, TATA-binding protein, TFIIB and TFIIF can form a quaternary complex in the absence of promoter DNA, indicating that a stable network of interactions exists between these proteins independent of promoter DNA. Single molecule studies can be used to learn how different modes of preinitiation complex assembly impact transcriptional activity.
HMGB1 (high mobility group box protein 1) is an architectural protein that facilitates formation of protein-DNA assemblies involved in transcription, recombination, DNA repair, and chromatin remodeling. Important to its function is the ability of HMGB1 to bend DNA non-sequence specifically. HMGB1 contains two HMG boxes that bind and bend DNA (the A box and the B box) and a C-terminal acidic tail. We investigated how these domains contribute to DNA bending by HMGB1 using single molecule FRET, which enabled us to resolve heterogeneous populations of bent and unbent DNA. We found that full length HMGB1 bent DNA more than the individual A and B boxes. Removing the C-terminal tail resulted in a protein that bent DNA to a greater extent than the full length protein. These data suggest that the A and B boxes simultaneously bind DNA in the absence of the C-terminal tail, but the tail modulates DNA binding and bending by one of the HMG boxes in the full length protein. Indeed, a construct composed of the B box and the C-terminal tail only bent DNA at higher protein concentrations. Moreover, in the context of the full length protein, mutating the A box such that it could not bend DNA resulted in a protein that bent DNA similarly to a single HMG box and only at higher protein concentrations. We propose a model in which the HMGB1 C-terminal tail serves as an intramolecular damper that modulates the interaction of the B box with DNA.
FRET (Forster resonance energy transfer) involves the transfer of energy from an excited donor fluorophore to an acceptor molecule in a manner that is dependent on the distance between the two. A biochemistry laboratory experiment is described that teaches students how to use FRET to evaluate distance changes in biological molecules. Students measured the apparent FRET between donor and acceptor fluorophores located on the ends of several DNAs of unknown lengths, enabling them to order the DNAs according to size. In addition, students investigated site-specific DNA cleavage by restriction endonucleases, using loss of apparent FRET to determine which enzyme cut sites were present in each of the DNAs. After completing this experiment, students understood the inverse relationship between changes in FRET and changes in distance, and understood how changes in FRET could be used to monitor a conformational change in a molecule. As an extension to the experiment, a tutorial is included that uses the same DNAs to illustrate the ability of singlemolecule FRET measurements to resolve heterogeneity in a sample, which cannot be done via more traditional ensemble measurements.
Thirty-five years after the discovery of MYC and despite intensive research effort, there is still little understanding of how this transcription factor acts or why it is an oncogene. Several models have recently emerged that cast doubt on the prevailing view of MYC as a gene-specific transcription factor and instead envision its oncogenic mechanism as a global amplifier or invader of enhancer regions. We seek to evaluate these hypotheses of MYC function and shed light on the in-vivo behavior of this transcription factor by determining how MYC modulates the kinetics of transcription events in single cells using single-molecule RNA fluorescence in-situ hybridization (RNA FISH) and live cell imaging of transcription. Our initial results testing the effect of MYC overexpression on an exogenous reporter gene in U2-OS cells shows that MYC increases the saturation level of the reporter dose response, indicating that MYC is able to act as a general amplifier without the need of enhancer regions. Unexpectedly however, the positive correlation between MYC and reporter RNA levels in the population is not reflected at the single cell level. This data suggests that static measurements of RNA abundance in single cells-such FISH or RNA-Seq-needs to be interpreted within a framework that explicitly takes into account the dynamic nature of transcription events. Further, as a way to directly assess the effect of the MYC protein on the transcription behavior of reporter genes, we engineered a photo-activatable version of MYC (PA-MYC) to achieve spatial and temporal control of the transcription factor within living cells. This shall allow us to follow the direct response of the transcription in response to MYC perturbation.
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