O. V. Chertkov

Preparation of Mononucleosomal Templates for Analysis of Transcription with RNA Polymerase using spFRET

Single positioned nucleosomes have been extensively employed as simple model experimental systems for analysis of various intranuclear processes. Here we describe an experimental system containing positioned mononucleosomes allowing transcription by various RNA polymerases. Each DNA template contains a pair of fluorescent labels (Cy3 and Cy5) allowing measuring relative distances between the neighboring coils of nucleosomal DNA using Forster resonance energy transfer (FRET). The single-particle FRET (spFRET) approach for analysis of DNA uncoiling from the histone octamer during transcription through chromatin is described in detail.

Development of fluorescently labeled mononucleosomes for the investigation of transcription mechanisms by single complex microscopy

Fluorescence microscopy of single molecules and complexes is an increasingly popular method for research on nucleosomes and functionally important processes involving these biological objects. Precisely positioned mononucleosomes have been developed in the present work using a fluorescently labeled DNA template; such nucleosomes are novel tools for the investigation of structural rearrangements of chromatin during transcription by RNA polymerase (RNAP). Two fluorophores, the donor Cy3 and the acceptor Cy5, were introduced into the nontranscribed DNA strand. DNA coiling around the histone octamer resulted in the positioning of both fluorophores on adjacent DNA coils in the middle part of the nucleosome. The distance between the fluorophores was less than 60 Å, and, therefore, Förster resonance energy transfer (FRET) could occur. Structural rearrangements in the nucleosomes were detected using the changes in FRET efficiency measured in fluorescence microscopic studies of individual complexes of nucleosomes with RNAP. Labeling had no effect on the ability of RNAP to transcribe DNA in nucleosomes. An open complex with RNAP and elongation complexes arrested in positions–39 and–5 relatively to the nucleosome border were obtained and characterized. More than 80% of the nucleosomes have been shown to retain their structure (that is, recover the initial positioning of DNA on the histone octamer) after the completion of transcription. The experimental system developed opens up new possibilities for research on nucleosome structure and its modulation by various protein chaperones and chromatin remodeling complexes.

Analysis of Nucleosome Transcription Using Single-Particle FRET

Many biological reactions including transcription of a gene are too complex and heterogeneous to be understood by studying ensembles of interacting molecules. In these cases analysis of single complexes can clarify structural and dynamic aspects of these processes. Here we report that single-particle Forster resonance energy transfer (spFRET ) microscopy is applicable to investigation of transcription through nucleosomes by an RNA polymerase . Mononucleosomes that support transcription were assembled from core histones and short DNA containing the T7A1 promoter and strong 603 nucleosome-positioning sequence. Fluorophores (Cy3 and Cy5) were introduced in the neighboring coils of nucleosome DNA in spatially close positions without disturbance of nucleosomal structure or transcription. Such labeling allows the changes in the Cy3–Cy5 distance caused by DNA uncoiling from the octamer or DNA looping to be monitored as changes in FRET efficiency. spFRET measurements for freely diffusing single nucleosomes were conducted using a laser scanning confocal microscope equipped with avalanche photodiodes. Nucleosome subpopulations that differ in FRET efficiency (i.e. in nucleosome structure) were revealed. RNA polymerase was stalled in distinct positions on the nucleosomal DNA during transcription, and the structures of these complexes were characterized with spFRET microscopy.

Molecular mechanisms of transcription through a nucleosome by RNA polymerase II

The RNA polymerase II (Pol II)-type mechanism is conserved from yeast to human. After transcription initiation, Pol II usually pauses upstream of or in a nucleosome within the early transcribed region of a gene. Then Pol II overcomes the initial nucleosomal barrier and efficiently proceeds through chromatin. At a low to moderate transcription rate, Pol II progression is only characterized by displacement or exchange of H2A/H2B dimers, and the resulting hexasomes (subnucleosomes lacking one of the H2A/H2B dimers) survive owing to the formation of small intranucleosomal DNA loops. The nucleosome structure is restored before the next Pol II complex starts transcription at a moderate transcription rate. The mechanism most likely helps to preserve the histone code during transcription. As the transcription rate increases, the distance between transcribing Pol II complexes becomes shorter, and trailing Pol II complexes may encounter the hexasome formed in the previous transcription round before the H2A/H2B dimer rebinds to it to restore a complete nucleosome. An unstable intermediate with fewer DNA-histone contacts forms in this case, resulting in the eviction or exchange of all core histones during transcription. Various protein factors and histone chaperones are involved in chromatin transcription by Pol II in vivo.

Analysis of Nucleosome Structure in Polyacrylamide Gel by the Förster Resonance Energy Transfer Method

A technique for analyzing the structure of (Cy3, Cy5)-labeled nucleosomes in polyacrylamide gel after electrophoresis under native conditions was developed based on the Förster resonance energy transfer (FRET) effect. It has been shown that the correct application of this technique requires monitoring of nonspecific intermolecular FRET and fluorescence reabsorption. A comparative analysis of the results of the FRET measurements of two types of nucleosomes and their complexes with yeast protein FACT was performed, which confirmed the similarity of the structural features of nucleosomes detected in the gel and in aqueous solution. Application of FRET analysis in combination with electrophoresis makes it possible not only to separate, visualize components of a complex mixture, and to evaluate their relative content but also to characterize the structural differences between these complexes in situ.

Molecular mechanisms of transcription through chromatin by RNA polymerase III: Part 2

Nucleosome forms a so-called nucleosome barrier, which is a serious difficulty for a transcriptional apparatus. We considered studies of formation and nucleosome barrier closing by some types of RNA polymerases. We showed that different polymerases use similar mechanisms for chromatin transcription. We present data testifying to the high similarity between transcriptional and ATP-dependent chromatin remodeling. We propose a nucleosome-mediated model of transcription.

Purification and concentration of RNA polymerase on Ni-lipid monolayers

Knowledge of the chromatin transcription intermediate structures can shed light on the functioning of RNA polymerase in the cell, which is important both for basic science and for the development of therapeutic approaches for the treatment of diseases associated with transcription disorders. In this study, we used affinity monolayers formed by lipids bound to Ni to concentrate RNA polymerase from a dilute solution containing glycerol. It has been shown that RNA polymerase can be isolated and concentrated on a Ni-lipid monolayer from solutions containing cryoprotectants, which usually make it difficult to visualize samples by electron microscopy. This method of isolation allowed us to obtain more diverse spatial orientations of molecules on carbon film, which facilitates three-dimensional reconstruction from TEM data. The results obtained can be used in further structural studies of the transcription processes to identify interactions in transcriptional complexes containing RNA polymerase.

Change in Linker DNA Conformation upon Histone H1.5 Binding to Nucleosome: Fluorescent Microscopy of Single Complexes

A method for fluorescently labeled DNA synthesis, which makes it possible to assemble mononucleosomes with 40 bp linkers, was developed. Cy3 and Cy5 labels were introduced into the linkers at distances of 10 bp before the first nucleotide and 15 bp after the last nucleotide of the nucleosome positioning DNA sequence, respectively. Without histone H1.5, f luorescence microscopy of single complexes revealed two equally probable states of nucleosomes. The states were different in the linker conformation: the open one with the energy transfer efficiency (E) between the labels of 0.06 and the closed one with E = 0.37, when the linkers are brought together. Binding of histone H1.5 with nucleosomes occurs in a range of nanomolar concentrations, and the complex formation rate is significantly higher as compared with its dissociation rate. The significant convergence of the DNA linkers (E = 0.73) is observed in these complexes together with the higher conformation uniformity in the region where the labels are localized. The developed nucleosomal constructs represent highly sensitive f luorescent sensors that can be used for the analysis of structural linker rearrangements. Also, in combination with microscopy of single complexes, they are suitable for studying the structure of complexes of nucleosomes with different chromatin architectural proteins.

Molecular mechanisms of chromatin transcription by RNA polymerase III. Part 1

Chromatin provides for the dense packing of DNA in eukaryotic cell nuclei and its proper functioning. The present review describes mechanisms of chromatin transcription by RNA polymerase III (RNAP III). This mechanism is characteristic of eukaryotic RNAP III, some bacteriophage RNAPs, and many ATP-dependent chromatin remodeling complexes. During the remodeling of chromatin according to this mechanism, nucleosomes move along the DNA molecule without octamer release into the liquid phase.

Single-Particle FRET Microscopy of Immobilized Nucleosomes: Technique Development

Mononucleosomes formed by histone octamer and short DNA is an advanced model system for investigation of RNA polymerase (RNAP) transcription and its modulation with various transcription factors. Recent achievements of fluorescent microscopy allow one to complement these studies with single-particle Forster resonance energy transfer (spFRET) analysis. FRET efficiency between Cy3 and Cy5 dyes introduced in the neighboring coils of nucleosomal DNA is a sensor of structural changes caused by DNA unwrapping or looping, or restoration of DNA-histone interactions. Here we report on experimental setup and protocols for spFRET microscopy of immobilized nucleosomes. Using confocal laser scanning and total internal reflection fluorescence microscopy we demonstrated preservation of the nucleosome structure during immobilization and long-term (>100 s, ca. 140 ms/frame) spFRET kinetic measurements. The effect of ionic strength on nucleosome structure was studied. Applications of spFRET microscopy of immobilized nucleosomes include analysis of their structural dynamics (DNA “breathing”), kinetics of formation/dissociation of DNA-protein complexes, formation and structure of stalled elongation complexes with RNAP, as well as conformational transitions in nucleosome structure in the course of transcription.