Abstract P-23: Histone N-terminal Tails Reduce Early Nucleosomal Pausing during Transcription

Background: Nucleosomes are the barriers to transcript elongation by RNA polymerase 2 (Pol 2) in vitro and in vivo. Formation and overcoming the barrier are important for transcription regulation. N-terminal tails of core histones do not affect the inner structure of nucleosomal core. However, strongly positively charged tails can interact with the DNA, thereby impeding polymerase progression through the template. Removal of histone tails was shown to facilitate transcription through a nucleosome by both yeast and human Pol 2, and the effect was most noticeable at lower ionic strength (40 mM KCl). In vivo experiments established a new mechanism of overcoming of +1 nucleosomal barrier by removal of histone tails by specific regulative proteinase. As +1 nucleosomal barrier is formed mostly by the promoter-proximal part of the nucleosomal DNA, here we address the effects of histone tails on elongation through this part of the nucleosome. Methods: We have studied the effect of histone tails on transcription by yeast Pol 2 and model enzyme E. coli RNA polymerase utilizing very similar mechanisms of elongation through chromatin. 603 nucleosomes were transcribed in vitro using purified proteins and components. To focus on the proximal part of the nucleosome, transcript elongation was conducted for a limited time and at low ionic strength. Results: For the phosphorylated form of yeast Pol 2 and E. coli RNAP, histone tail removal significantly reduces the strong nucleosome-specific pausing that the yeast polymerase encounters ∼15 bp within the 603 nucleosome and further downstream, leading to both increased traversal of the pause and the accumulation of complexes paused at more distal locations. However, tail removal did not lead to a significant increase in full traversal of either nucleosomal template. The effect of histone tails removal was cognate for both enzymes but differs in detailed effect on the barrier. Conclusion: Histone tails provide a significant part of the nucleosomal barrier to transcript elongation by Pol 2-type mechanism. The effect is very pronounced in the promoter-proximal part of the nucleosomal DNA, suggesting that histone tails could play a role during the regulation of the +1 nucleosomal barrier. The role of Pol 2 CTD phosphorylation and formation of the intranucleosomal loops in the regulation of +1 nucleosomal barrier will also be addressed

Structure of an Intranucleosomal DNA Loop That Senses DNA Damage during Transcription

Transcription through chromatin by RNA polymerase II (Pol II) is accompanied by the formation of small intranucleosomal DNA loops containing the enzyme (i-loops) that are involved in survival of core histones on the DNA and arrest of Pol II during the transcription of damaged DNA. However, the structures of i-loops have not been determined. Here, the structures of the intermediates formed during transcription through a nucleosome containing intact or damaged DNA were studied using biochemical approaches and electron microscopy. After RNA polymerase reaches position +24 from the nucleosomal boundary, the enzyme can backtrack to position +20, where DNA behind the enzyme recoils on the surface of the histone octamer, forming an i-loop that locks Pol II in the arrested state. Since the i-loop is formed more efficiently in the presence of SSBs positioned behind the transcribing enzyme, the loop could play a role in the transcription-coupled repair of DNA damage hidden in the chromatin structure

Role of Histone Tails and Single Strand DNA Breaks in Nucleosomal Arrest of RNA Polymerase

Transcription through nucleosomes by RNA polymerases (RNAP) is accompanied by formation of small intranucleosomal DNA loops (i-loops). The i-loops form more efficiently in the presence of single-strand breaks or gaps in a non-template DNA strand (NT-SSBs) and induce arrest of transcribing RNAP, thus allowing detection of NT-SSBs by the enzyme. Here we examined the role of histone tails and extranucleosomal NT-SSBs in i-loop formation and arrest of RNAP during transcription of promoter-proximal region of nucleosomal DNA. NT-SSBs present in linker DNA induce arrest of RNAP +1 to +15 bp in the nucleosome, suggesting formation of the i-loops; the arrest is more efficient in the presence of the histone tails. Consistently, DNA footprinting reveals formation of an i-loop after stalling RNAP at the position +2 and backtracking to position +1. The data suggest that histone tails and NT-SSBs present in linker DNA strongly facilitate formation of the i-loops during transcription through the promoter-proximal region of nucleosomal DNA

Behavioural Features of Various Social Groups on the Internet

Objective: The objective of the article is to find an effective model for teaching children using modern educational technologies.Background: The relevance of the study is that the formation of communication in social networks is determined by the ability to search for strategies for social contacts. In this regard, a significant part of people uses a ready-made communicative model created on a technological basis in social networks. The issue of understanding the general structure of communication in the formation of the social structure of a person in adolescents remains debatable.Method: The article notes that the operation of social networks is governed by purely technical methods and technologies, which can be considered as prerequisites for the translation of such technologies into the space of social interactions.Results: The authors show that the possibility of a regulatory impact on adolescent behaviour fully meets the principles of organising network communities with an orientation on each person's individual characteristics. Conclusion: The use of Internet technologies allows for the growth and timely development of communication technologies for the formation of a balanced personality in a globalised world

Role of Histone Tails and Single Strand DNA Breaks in Nucleosomal Arrest of RNA Polymerase

Transcription through nucleosomes by RNA polymerases (RNAP) is accompanied by formation of small intranucleosomal DNA loops (i-loops). The i-loops form more efficiently in the presence of single-strand breaks or gaps in a non-template DNA strand (NT-SSBs) and induce arrest of transcribing RNAP, thus allowing detection of NT-SSBs by the enzyme. Here we examined the role of histone tails and extranucleosomal NT-SSBs in i-loop formation and arrest of RNAP during transcription of promoter-proximal region of nucleosomal DNA. NT-SSBs present in linker DNA induce arrest of RNAP +1 to +15 bp in the nucleosome, suggesting formation of the i-loops; the arrest is more efficient in the presence of the histone tails. Consistently, DNA footprinting reveals formation of an i-loop after stalling RNAP at the position +2 and backtracking to position +1. The data suggest that histone tails and NT-SSBs present in linker DNA strongly facilitate formation of the i-loops during transcription through the promoter-proximal region of nucleosomal DNA

Seedling Biometry of <i>nud</i> Knockout and <i>win1</i> Knockout Barley Lines under Ionizing Radiation

The genes NUD and WIN1 play a regulatory role in cuticle organization in barley. A knockout (KO) of each gene may alter plant mechanisms of adaptation to adverse environmental conditions. A putative pleiotropic effect of NUD or WIN1 gene mutations in barley can be assessed in a series of experiments in the presence or absence of a provoking factor. Ionizing radiation is widely used in research as a provoking factor for quantifying adaptive potential of living organisms. Our aim was to evaluate initial stages of growth and development of barley lines with a KO of NUD or WIN1 under radiation stress. Air-dried barley grains with different KOs and wild-type control (WT) were exposed to γ-radiation at 50, 100, or 200 Gy at a dose rate of 0.74 R/min. Approximately 30 physiological parameters were evaluated, combined into groups: (1) viability, (2) radiosensitivity, and (3) mutability of barley seed progeny. Seed germination, seedling survival, and shoot length were similar among all barley lines. Naked nud KO lines showed lower weights of seeds, roots, and seedlings and shorter root length as compared to win1 KO lines. The shoot-to-root length ratio of nud KO lines’ seedlings exceeded that of win1 KO and WT lines. In terms of the number of seedlings with leaves, all the KO lines were more sensitive to pre-sowing γ-irradiation. Meanwhile, the radioresistance of nud KO lines (50% growth reduction dose [RD50] = 318–356 Gy) and WT plants (RD50 = 414 Gy) judging by seedling weight was higher than that of win1 KO lines (RD50 = 201–300 Gy). Resistance of nud KO lines to radiation was also demonstrated by means of root length (RD50 = 202–254 Gy) and the shoot-to-root length ratio. WT seedlings had the fewest morphological anomalies. In nud KO lines, mainly alterations of root shape were found, whereas in win1 KO lines, changes in the color and shape of leaves were noted. Thus, seedlings of nud KO lines are characterized mainly by changes in the root system (root length, root number, and root anomalies). For win1 KO lines, other parameters are sensitive (shoot length and alterations of leaf shape and color). These data may indicate a pleiotropic effect of genes NUD and WIN1 in barley

Structure of an Intranucleosomal DNA Loop That Senses DNA Damage during Transcription

Transcription through chromatin by RNA polymerase II (Pol II) is accompanied by the formation of small intranucleosomal DNA loops containing the enzyme (i-loops) that are involved in survival of core histones on the DNA and arrest of Pol II during the transcription of damaged DNA. However, the structures of i-loops have not been determined. Here, the structures of the intermediates formed during transcription through a nucleosome containing intact or damaged DNA were studied using biochemical approaches and electron microscopy. After RNA polymerase reaches position +24 from the nucleosomal boundary, the enzyme can backtrack to position +20, where DNA behind the enzyme recoils on the surface of the histone octamer, forming an i-loop that locks Pol II in the arrested state. Since the i-loop is formed more efficiently in the presence of SSBs positioned behind the transcribing enzyme, the loop could play a role in the transcription-coupled repair of DNA damage hidden in the chromatin structure

Stabilization of Nucleosomes by Histone Tails and by FACT Revealed by spFRET Microscopy

A correct chromatin structure is important for cell viability and is tightly regulated by numerous factors. Human protein complex FACT (facilitates chromatin transcription) is an essential factor involved in chromatin transcription and cancer development. Here FACT-dependent changes in the structure of single nucleosomes were studied with single-particle Förster resonance energy transfer (spFRET) microscopy using nucleosomes labeled with a donor-acceptor pair of fluorophores, which were attached to the adjacent gyres of DNA near the contact between H2A-H2B dimers. Human FACT and its version without the C-terminal domain (CTD) and the high mobility group (HMG) domain of the structure-specific recognition protein 1 (SSRP1) subunit did not change the structure of the nucleosomes, while FACT without the acidic C-terminal domains of the suppressor of Ty 16 (Spt16) and the SSRP1 subunits caused nucleosome aggregation. Proteolytic removal of histone tails significantly disturbed the nucleosome structure, inducing partial unwrapping of nucleosomal DNA. Human FACT reduced DNA unwrapping and stabilized the structure of tailless nucleosomes. CTD and/or HMG domains of SSRP1 are required for this FACT activity. In contrast, previously it has been shown that yeast FACT unfolds (reorganizes) nucleosomes using the CTD domain of SSRP1-like Pol I-binding protein 3 subunit (Pob3). Thus, yeast and human FACT complexes likely utilize the same domains for nucleosome reorganization and stabilization, respectively, and these processes are mechanistically similar

<i>Lycaena helle</i> samples used in the study.

<p><i>Lycaena helle</i> samples used in the study.</p

Spatial genetic structure of <i>O</i>. <i>decorus</i> inferred using fastStructure with <i>k</i> = 2 and simple priors.

<p>Colours denote the two different genetic groups supported by a previous study relying on mtDNA markers [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151651#pone.0151651.ref022" target="_blank">22</a>].</p