Single-molecule FRET method to investigate the dynamics of transcription elongation through the nucleosome by RNA polymerase II

Abstract

Transcription elongation through the nucleosome is a precisely coordinated activity to ensure timely production of RNA and accurate regulation of co-transcriptional histone modifications. Nucleosomes actively participate in transcription regulation at various levels and impose physical barriers to RNA polymerase II (RNAPII) during transcription elongation. Despite its high significance, the detailed dynamics of how RNAPII translocates along nucleosomal DNA during transcription elongation and how the nucleosome structure dynamically conforms to the changes necessary for RNAPII progression remain poorly understood. Transcription elongation through the nucleosome is a complex process and investigating the changes of the nucleosome structure during this process by ensemble measurements is daunting. This is because it is nearly impossible to synchronize elongation complexes within a nucleosome or a sub-nucleosome to a designated location at a high enough efficiency for desired sample homogeneity. Here we review our recently developed single-molecule FRET experimental system and method that has fulfilled this deficiency. With our method, one can follow the changes in the structure of individual nucleosomes during transcription elongation. We demonstrated that this method enables the detailed measurements of the kinetics of transcription elongation through the nucleosome and its regulation by a transcription factor, which can be easily extended to investigations of the roles of environmental variables and histone post-translational modifications in regulating transcription elongation.

Structural Basis of Nucleosome Recognition and Modulation

“…[ 8 ] More recently, cryo‐electron microscopy (cryo‐EM) has become extremely powerful, and has had notable success in the structural characterization of nucleosomes and several nucleosome‐bound complexes, as has been reviewed in the past. [ 9–12 ] Significant contributions to our understanding of these complex machineries and their dynamics have also come from the complementary biophysical techniques such as nuclear magnetic resonance (NMR) spectroscopy, [ 13–16 ] Förster resonance energy transfer (FRET), [ 17–19 ] small‐angle X‐ray scattering (SAXS), [ 15,20–23 ] small‐angle neutron scattering, [ 23 ] native electro‐spray ionization mass spectrometry, [ 21 ] size‐exclusion chromatography—multi‐angle light scattering (SEC‐MALS), [ 24,25 ] analytical ultra centrifugation (AUC), [ 16,24,26 ] and hydroxyl radical footprinting, [ 21,27 ] along with Molecular Dynamics (MD) simulation, modeling, and docking studies. [ 15–17,23,27 ] The different chromatin factors and viral proteins use specific features on the nucleosome such as the nucleosomal acidic patch, specific super‐helical locations, PTM marks, and the histone tails to interact with and modulate the nucleosome.…”

PCNA monoubiquitination is regulated by diffusion of Rad6/Rad18 complexes along RPA filaments

“…smFRET measurements. Quartz microscope slides (Finkenbeiner, USA) were thoroughly cleaned as previously described 43 and slide surfaces were coated with polyethyleneglycol (PEG) and…”

“…was injected. After a 10 minute incubation to deplete oxygen, two-color smFRET measurements were performed using a prism-coupled total internal reflection fluorescence (TIRF) microscope system that is based on a Nikon TE2000 microscope (Nikon, Japan) as previously described 43 .…”

Optimization of nucleosome assembly from histones and model DNAs and estimation of the reconstitution efficiency

“…fying the influence of nucleosomal organization on the efficiency of DNA-dependent processes and demonstrating the importance of nucleosomal DNA context to both the nucleosome organization and the process efficacy (1)(2)(3). The performance of such investigations implicates the usage of reconstituted nucleosomes with the defined DNA sequence.…”

“…The primary unit of DNA compaction is nucleosome. Thus, to perform any DNA-dependent process such as replication, repair or transcription it is necessary to affect the chromatin context in whole and to nucleosomes in particular (1)(2)(3). There are multiple studies testi- ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell.…”