Short-Read Single-Molecule DNA Sequencing for Highly Parallel Analysis of Protein-DNA Interactions
Biophysical Journal Elsevier 114:3 (2018) 92a
Wide-Field Monitoring of Single Fluorescent Molecules and Nanoparticles without Immobilization
Biophysical Journal Elsevier 114:3 (2018) 169a
Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes
Nucleic Acids Research 46:2 (2018) 677-688
Abstract:
© The Author(s) 2017. Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex sub-sequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia colitranscription open complexes. Our results revealed the existence of two stable states within RNAP-DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.Single-molecule and super-resolution imaging of transcription in living bacteria.
Methods (San Diego, Calif.) 120 (2017) 103-114
Abstract:
In vivo single-molecule and super-resolution techniques are transforming our ability to study transcription as it takes place in its native environment in living cells. This review will detail the methods for imaging single molecules in cells, and the data-analysis tools which can be used to extract quantitative information on the spatial organization, mobility, and kinetics of the transcription machinery from these experiments. Furthermore, we will highlight studies which have applied these techniques to shed new light on bacterial transcription.Tracking Low-Copy Transcription Factors in Living Bacteria: The Case of the lac Repressor.
Biophysical journal 112:7 (2017) 1316-1327