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.

Keywords: Nucleosome; RNA polymerase II; Single-molecule FRET; Transcription elongation.

Figure 1.. The schematics of the single-molecule FRET system to investigate the dynamics of the nucleosome structure during transcription elongation.

(A) Nucleosome assembled on the shown template is complexed with RNAPII (yeast Pol II). Rpb1 CTD antibody is immobilized on a microscope slide surface via biotinylated Protein A that is conjugated to streptavidin coated on the slide. A FRET pair (Cy3 and Atto647N) is labeled at the +34^th and +112^th nucleotide. This FRET pair location is sensitive to the nucleosomal dynamics at the proximal-dimer/DNA contact region. (B) The sequence of the upper and lower strands of the transcription template illustrated in A.

Figure 2.. A schematic description of reaction chamber fabrication.

(A) A carved Parafilm® is placed on a microscope slide with 10 holes for sample injection and washing. A clean coverslip is placed on top of the film layer and the sandwich is subsequently heated at 110 °C for 90 seconds to fabricate 5 reaction chambers. (B) The slide reaction chamber is place upside down on an inverted microscope to monitor the surface immobilized elongation complexes.

Figure 3.. Fluorescence excitation source, optics, and their alignment.

Detailed description can be found in the main text. The objective must have a long enough working distance to ensure optical access to the elongation complexes immobilized upside down on the slide surface. Our setup is equipped with Nikon CFI Plan APO VC 60x water immersion objective (NA = 1.2, working distance 0.28 – 0.31 mm).

Figure 4.. Imaging optics alignment and the process of data collection and analysis.

(A) Imaging optics alignment. A fluorescence image out of the side port of an inverted microscope is passed through a width-adjustable slit. The image is split into two colors (Cy3 and Atto647N) with a 650 nm dichroic mirror. Each image is reformed by a relay lens on another conjugate image plane that is located at the CCD camera. (B) The reformed two-color images at the CCD camera are spatially separate (left: Cy3, right: Atto647N). A typical image is shown in figure S3. A time series of the images are taken and recorded as a movie. (C) On each movie frame, all pairs of Cy3 and Atto647N spots are identified. Each FRET pair at each movie frame gives the fluorescence intensities of the donor and the acceptor. A series of the fluorescence intensities plotted along time makes fluorescence intensity time trajectories for the donor and acceptor (C-1), which is used to generate a FRET time trajectory (C-2). C-3 shows a computer-simulated example of how data looks. (D) An illustration to show how a FRET state lifetime is measured. Detailed descriptions can be found in the main text.

Figure 5.. A typical smFRET time trajectory and a FRET transition histogram during transcription elongation.

(A) A typical smFRET time trajectory reveals pauses and elongation between pauses. Detailed description can be found in the main text. (B) FRET transition histogram obtained from 659 FRET transitions. “H”, “M”, and “L” FRET states represents high-, mid-, and low-FRET states, respectively. Further details can be found in the main text.