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Lecture 9, part 4 of 4
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IV. TRANSCRIPTIONALLY ACTIVE CHROMATIN

Problem: If DNA is complexed with proteins, won't that make it hard for transcription to occur?
The enzymes of the euk. transcriptional apparatus are adapted to the presence of chromatin proteins. This is the price euk. cells pay for having such large genomes. You can't have a large genome without having the extra overhead of organizing it, which is done by the chromatin proteins. Like everything else in eukaryotic cells, gene expression and replication are very deliberate and carefully orchestrated processes. As we will see, they are not simply diffusion-driven.

A. For transcription to occur, the chromatin structure of a gene must be 'open', that is, accessible to the transcriptional machinery.

How do we detect "open" chromatin structure?

    We can compare chromatin structure around the beta-globin gene of chick embryo erythroblasts (precursors to red blood cells), which do express globin genes, and an undifferentiated cell line MSB, which does not express globin genes. In erythroblasts, the DNA of the globin gene is preferrentially sensitive to DNAseI digestion. This suggests that the structure of the chromatin is looser in cells expressing globin.


In the autoradiogram, we see that even at the highest DNAseI concentration, the 4.6kb fragment is relatively insensitive to digestion in nuclei from a cell line that does not express the globin gene. In the erythroblasts, however, specific degredation of this sequence can be seen to occur even at the lowest concentration. The interpretation of this is that the globin gene is in a more open chromatin configuration, which provides greater access to DNAseI. This phenomenon is referred to as general nuclease sensitivity, because the whole gene appears to be digested. However, more detailed studies of certain genes can detect specific sites within transcriptionally active genes that are hypersensitive to nuclease digestion.
From Lodish et al. Molecualr Cell Biology
http://www.ncbi.nlm.nih.gov/books/bookres.fcgi/mcb/ch9f32b.gif




M is a marker lane, with a mixture of DNAs generated by digesting this gene with either BamH1, Xba1, Pst1 or Alu1, and then mixing all of these DNAs together.Now, when the DNAse-ed DNA is digested with Hind3, the +1 to +210 probe will only detect fragments upstream from the Hind3 site. If we do a limiting digestion with DNAseI, in any given fragment, only one of several possible hypersensitive sites will be digested in a given molecule. Thus, we get a "ladder", in which each rung represents a different cut site. Note that there are two constitutive hypersensitive sites, even in uninduced (aerobic) nuclei, whereas in induced nuclei, we see 8 distinct hypersensitive sites.

from Anna-Lisa Paul, Vimla Vasil, Indra K. Vasil, and Robert J. Ferl. Constitutive and anaerobically induced DNase-I-hypersensitive sites in the 5′ region of the maize Adh1 gene. PNAS 84:799-803

B. Transcriptionally active DNA remains associated with histones, although nucleosome configuration may be different in active vs. inactive chromatin.

a. DNA seems to be complexed in nucleosomes on both sides of an RNA polymerase.

"BEADS ON A STRING" WITH NASCENT RNA TRANSCRIPTS [Alberts,  Mol. Biol. of the Cell, 2nd Ed., p410, Fig. 8-37]


Electron micrograph shows nucleosome structure on both sides of an RNA poymerase II transcription complex.

2. Nucleosome structure "opens up" during transcription, in a process called "chromatin remodeling"

Chromatin remodeling: insights and intrigue from single-molecule studies
Bradley R Cairns. Nature Structural & Molecular Biology 14, 989 - 996 (2007) Published online: 5 November 2007
doi:10.1038/nsmb1333

The precise details of chromatin remodeling are still under investigation. However, the figure below illustrates one model for chromatin remodeling. In (d), D and Tr domains of the ocatmer "walk" along the double helix by rotating around the Hinge domain. Thus, while the DNA is more accessible to the solvent, and hence, more sensitive to nucleases, the nucleosome never completely dissociates from the DNA helix.



Image displayed by hypertext link to
http://www.nature.com/nsmb/journal/v14/n11/images/nsmb1333-F3.gif