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The FACT (facilitates chromatin transcription) complex is required for transcript elongation through nucleosomes by RNA polymerase II (Pol II) in vitro. Here, we show that FACT facilitates Pol II-driven transcription by destabilizing nucleosomal structure so that one histone H2A-H2B dimer is removed during enzyme passage. We also demonstrate that FACT(More)
Nucleosomes, the nucleohistone subunits of chromatin, are present on transcribed eukaryotic genes but do not prevent transcription. It is shown here that the large yeast RNA polymerase III transcribes through a single nucleosome. This takes place through a direct internal nucleosome transfer in which histones never leave the DNA template. During this(More)
Transcription of eukaryotic genes by RNA polymerase II (Pol II) is typically accompanied by nucleosome survival and minimal exchange of histones H3 and H4. The mechanism of nucleosome survival and recovery of chromatin structure remains obscure. Here we show how transcription through chromatin by Pol II is uniquely coupled with nucleosome survival.(More)
The mechanism by which nucleosome cores are displaced and re-formed during transcription in vitro has been investigated. A nucleosome core was assembled on a short linear DNA template (227 bp) containing an SP6 RNA polymerase promoter and a nucleosome-positioning sequence. Transcription induced the translocation of the nucleosome core over 75 or 80 bp to(More)
We have studied the kinetics of transcription through a nucleosome core. RNA polymerase transcribes the first approximately 25 bp of nucleosomal DNA rapidly, but then hits a barrier and continues slowly to the nucleosomal dyad region. Here, the barrier disappears and the transcript is completed at a rapid rate, as if on free DNA, indicating that histone(More)
In eukaryotic genomes, nucleosomes function to compact DNA and to regulate access to it both by simple physical occlusion and by providing the substrate for numerous covalent epigenetic tags. While competition with other DNA-binding factors and action of chromatin remodeling enzymes significantly affect nucleosome formation in vivo, nucleosome positions in(More)
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(More)
Maintenance of nucleosomal structure in the cell nuclei is essential for cell viability, regulation of gene expression and normal aging. Our previous data identified a key intermediate (a small intranucleosomal DNA loop, Ø-loop) that is likely required for nucleosome survival during transcription by RNA polymerase II (Pol II) through chromatin, and(More)
Nucleosomes uniquely positioned on high-affinity DNA sequences present a polar barrier to transcription by human and yeast RNA polymerase II (Pol II). In one transcriptional orientation, these nucleosomes provide a strong, factor- and salt-insensitive barrier at the entry into the H3/H4 tetramer that can be recapitulated without H2A/H2B dimers. The same(More)
Chromatin insulators block the action of transcriptional enhancers when interposed between an enhancer and a promoter. In this study, we examined the role of chromatin loops formed by two unrelated insulators, gypsy and Fab-7, in their enhancer-blocking activity. To test for this activity, we selected the white reporter gene that is activated by the(More)