The goal of this line of research is to investigate the assembly mechanism, dynamics, and heterogeneity of human transcription factor/DNA complexes, and how these parameters contribute to transcriptional control. To do so, we are leveraging the unique abilities of the techniques of single-molecule fluorescence co-localization and single-molecule fluorescence resonance energy transfer (smFRET), which together can provide unprecedented insight into the biological function of nucleoprotein complexes. Single-molecule experiments complement the knowledge gained from ensemble biochemical experiments by allowing the observation of sub-populations of molecules that exist in distinct states and also the measurement of dynamic behavior in individual molecules, which are obscured by averaging across all the molecules present in an ensemble. We are using single molecule approaches to investigate the assembly and activity of complexes containing promoter DNA, transcriptional regulatory proteins, general transcription factors, and RNA polymerase II. To this end, we maintain a shared objective-based TIRF (total internal reflection fluorescence) microscope and have a home-written suite of software for data analysis.

We have used smFRET to study DNA bending by the TATA binding protein and the architectural protein HMGB1.

smFRET data collection and analysis

We use single molecule fluorescence colocalization to study human RNA polymerase II transcription.

Visualization of transcription by TIRF

Relevant research publications:

  • Ly, E., Powell, A.E., Goodrich, J.A., Kugel, J.F. (2020). Release of human TFIIB from actively transcribing complexes is triggered upon synthesis of 7 nt and 9 nt RNAs. J. Mol. Biol. 
  • Blair, R.H., Horn, A.E., Pazhani, Y., Grado, L., Goodrich, J.A., and Kugel, J.F.  (2016).  The HMGB1 C-Terminal Tail Regulates DNA Bending.  J. Mol. Biol.  428: 4060-4072.
  • Horn, A.E., Kugel, J.F., and Goodrich, J.A.  (2016).  Single molecule microscopy reveals mechanistic insight into RNA polymerase II preinitiation complex assembly and transcriptional activity.  Nucl. Acids Res.  44: 7132-7143.
  • Blair, R.H. Goodrich, J.A., and Kugel, J.F.  (2012).  Single molecule FRET shows uniformity in TBP-induced DNA bending and heterogeneity in bending kinetics.  Biochemistry.  51:7444-7455.
  • Gilman, B., Drullinger, L.F., Kugel, J.F., and Goodrich, J.A.  (2009).  TATA binding protein and transcription factor IIB induce transcript slipping during early transcription by RNA polymerase II.  J. Biol. Chem.  284: 9093-9098.
  • Hieb, A.R., Halsey, W.A., Betterton, M., Perkins, T., Kugel, J.F., and Goodrich, J.A.  (2007).  TFIIA changes the conformation of the DNA in TBP/TATA complexes and increases their kinetic stability.  J. Mol. Biol.  372: 619-632.
  • Hieb, A.R., Baran, S., Goodrich, J.A., and Kugel, J.F.  (2006).  An 8 nt RNA triggers a rate-limiting shift of RNA polymerase II complexes into elongation.  EMBO J.  25: 3100-3109.