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24 Cards in this Set

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Implications of DNA being much longer than the cell
1 meter of DNA --> micrometer magnitude of nucleus' diameter. This is not hard to image - that it can be so tightly coiled. But what is hard ti imagine is that how it is able to function with this setup. How can enzymes find their way to this condensed material? How can polymerases find TATA boxes, and activators, their UAS?
Metaphase Chromosome structure
The metaphase chromosome contains condensed scaffold associated chromatin. This chromatin is made up of 30 nanometer diameter fibers of packed nucleosomes from the 11 nm or the beads on a string structure. 6 nucleosomes per turn of the 11 nm (active - transcription takes place, polymerase can move along chromatin) fiber makes the 30 nm fiber (inactive).
11 nm fiber
- this fiber is isolated in low ionic strength buffer (low salt)
- beads on a string idea
- possibly, the active form
- may form from the 30 nm when 30nm is acted upon by histone Deactylases (HDAC)
30 nm fiber
-Isolated in physiological ionic strength buffer (isotonic to cellular salt concentration - suggests that this is what the chromatin structure is usually like?)
- possibly the inactive form
- may form from 11nm when acted upon by Histone Acetyl Transferases (HAT)
Nucleosome structure
- nucleosomes are not merely beads on a string - they are a means of wrapping negatively charged DNA (because of Phosphate backbone), which is 2nm in diameter) around positively charged spools)
- Histone octameric core is an octamer consisting of 2 molecules each of H2A, H2B, H3, and H4
- DNA wraps around histone octameric core via 2 turns - this means that there is an entry point and exit point of dna
- very little is known about H1 histone's function because we can't do genetics in humans because of long gestation and generation period. So we study using model organisms like drosophilla, yeast (quick replication time), C. Elegans, arabadopsis, mouse
Histone Tails
- nucleosomes are not merely beads or spools
- Amino terminal tails of histones extend from core.
- can't be seen in x ray crystallography because you need proteins (in high concentration) to form a crystalline array (when they all fold the same way) - when structures extend, you can't form crystalline array.
- Therefore, the way that the histone tails were found was by adding protease (like trypsin or chymotrypsin) to chromatin and running it on a gel) - only the tails were missing - this is evidence that the tails extend because only they were missing and not the other parts of the histone octameric core because the core is not accessible to protease.
Histones and their acetylation (and how is acetylation detected)
- lysines in the histone tails are acetylated (at the N terminus end)
- only lysines are acetylated.
- it was found that cell lines in which transcription was active had a lot of acetylation. acetylation is detected by feeding cell labeled acetate - it takes up acetate, turns it into acetyl coa, which donates acetyl group to lysine - so acetylated proteins are labeled with labeled acetate.
- a post-translational modifiction like methylation, phosphorylation, ubiquitination, etc.
- rapid and reversible.
- neutralizes postivie charge on lysines - this affects electrostatic interactions - histone tails can't interact with negative charged DNA as much - exposing them to enzymes
- Post translational modifications (reversible) are important because they allow the cell to change the function of proteins, and enzyems. This is needed because you wouldn't want a protein that is involved in transcription to always be involved in transcription (otherwise, the gene would always be on).
- Histone Acetyl Transferases (HAT) and Histone Deacetylases (HDAC) mediate acetylation
- it is just one way of gene activation and repression, but it is common.
- acetylation (and deactylation) are pharmaceutically important because giving HAT and HDAC inhibitors can allow for increased or decrased gene activity.
How does Acetylation activates genes?
- upstream of genes is the UAS, or upstream activator sequence. This always has proteins bound to it (like the GAL4, an activator protein which is always bound to UAS, but environmental factors can cause more GAL 4 to bind, increasing gene activity). UAS is sensitive to nucleases.
- acetylation is thought to open up chromatin structure (at each gene) via the following mechanism.
1. UAS binds gnc4, which has a DNA binding domain as well as an activation domain, which recruits many proteins including the Mediator complex of several proteins, and an HAT, gnc5. This gnc5 acetylates 1 or 2 nearby nucleosomes on either side, lowering the affinity of the histone tails 16-24 for the negatively charged faces of H2 - causing the chromatin structure to open up. This ALLOWS for transcription (increased gene activity) because RNA polymerase can now access the TATA box and begin transcription (but it doesn't cause it, which is done by RNAP)
Deactylation and gene repression
- URS - upstream repressor sequence
- binds ___6 (look it up on pubmed), which very much like gnc4 has DNA binding domain, but also repressor domain, which recruits rpd3 (among dozens of other proteins, which may just stabilize rpd3's catalytic activity), which very much like GNC5 acts of nearby nuclesomes, by deactylating them - this causes for an increase in affinity for the histone tails to neigbhoring histones, causing the gene to go from 11 to 30 nm structure.
What is the evidence that acetylated chromatin (in which gene is active) is more open?
- human RBCs don't have nucleus.
- but bird RBCs (normal ones) do, and they produce globin, a 4.6kb gene.
- globin has two bam h1 flanking it.
- when gene is in 30nm form, the globin gene is tightly bound and when in 11 nm form, it is not. Therefore DNAse can chop up globin only in 11nm form.
- MSB cells in bird cannot produce globin.
- Therefore in normal avian RBCs, at high enough concentration of DNAse (like .05 ug/ml), when you do a Southern blot, globin is not present at 4.6kb because it has been chopped up because it was in the acetylated 11nm form (so that the gene can be active and the globin protein can be produced).
- As DNAse concentration increases, the globin band from normal avian RBCs got fainter and fainter. At 1.5 ug/ml, no band. But at the same concentration for MSB cells, band is there because they don't need to produce globin - the gene is in 30nm form, (BAMHI can still access the sites), but DNAse can't chop the gene up, so a band at 4.6 kb is present.
How do UAS binding proteins bind to UAS if they are bound to histones
- UAS binding proteins would have to have more affinity to UAS than histones, so they may be able to move histones out of the way
- it is like a competition between UASbp and histones.
Peterson Experiment
- to understand role of k16-ac in higher order folding of nucleosomes
- experimental setup - designed 2 sets of 12 arrays of nuclosomes. One set had everything deactylated, while the other set had acetylations only at k16-ac.
- there are four constructs: wild type (deactylated?)histone, R23C (which is a control for the ligated protein fragements; essentially same as WT), K16AC , which is only acetylated at H4 K16, and N terminal deletion.
- without MgCL2, all four has same S, around 32 because high salt is needed for higher order folding
- but with MgCl2, WT and R23C has same phenotype, with S around 50, and K16AC and Delta N had same phenotype with S around 40. This shows that K16AC is needed for higher order folding and that only that residue is needed for going to 30 nm structure since Delta N had the same phenotype.
- S depends on mass and shape.
- what are the other sites for?
H4 residues 16-24
- contain lots of positive charges
- they interact BETWEEN histones ; the positive charges of H4 interact with the negatively charged face of H2 (but bottom of p.427 mentions that tail interacts with DNA, and not other histones).
- 16-24 is not really part of the amino terminal tail; it is in the region where the tail just begins - it is hard for the enzymes to access it.
Aberrant histone acetylation
- associated with APL, Huntington's disease, etc.
Metaphase chromomse structure
p. 428, fig 10-24.
- Metaphse chromosome is more condensed than interphase chromosome. Interphase chromosome involves a chromosome scaffold with which the 30nm fibers associated, which are the 11 nm fibers with 6 nucleosomes per turn, which have DNA molecules (2nm in diameter) coil twice around them.
Non histone proteins associated with Chromosomes
- the chromosome scaffold: when detergent gets rid of histones and denatures DNA, the scaffold, which resembles shape of metaphase chromosome, still exists. It attaches the 30nm fibers every 1-4 million base pairs (Fig.10-25) - physical distance, ascertained by fluorescent probes is smaller than that of the base pair distance.
- Transcription factors
- High mobility group proteins.
Chromosome banding pattern; polytene chromosomes
- stains bind to chromatin differentially; makes it easy to identify chromosomes
- polytene chromosomes - in salivary glands of larval Drosophilla, cells are big, so chromosome duplicates with creating daughter cells - this amplifies gene copy.
Heterochromatin and euchromatin
- heterochromatin stains more darkly.
- most transcription occurs in euchromatin and nucleolus.
- but heterochromatin contains some transcriptionally active genes also.
- and euchromtin also contains inactive and nontranscribed regions of DNA.
Features of a chromosome; four necessary elements
1. Single DNA molecule
2. Replicates in S phase
3. Segregatesi in a stable manner (fidelity) --> when daughter cell is made, it needs exactl the same chromosomes
- Needs the following 4 elements:
1. ARS (Autonomic Replications Sequence)
2. Centromeric Sequence
3. Telomeric Sequences
4. Size (atleast 50kb DNA)
- Autonomous Replication Sequence - contains Origin of Replication: it has the sequences that replication apparatus recognizes to proceed (usually) with bidirectional replication
- without it, chromosome can't replicate
- with it, but without centromere, chromosomes can replicate, but can't segregate properly - thus only 5-20% cell have plasmid (ascertained by growing cells in leu+ medium to see how many grow)
- sequence of about 120 bp
- is recognized by Centromere Binding Factor 1 and Centromere binding factor 3 (complex of 4 proteins) that recruits microtubules
- with centrome and ars, cells can replicate and segregate properly, but still only 90% of cell have plasmid (as opposed to 1 in a million not having it).
- kinetochore is the centromere sequence
- centromere is not needed for replication, but needed for segregation
- linear sequences without telomeres are not stable (even if they have centromere and ARS)
- telomere is not needed for replication or segregation - they are present to protect ends and allow them to replicate/ segregate properly.
- small linear chromosomes require at least 50kb DNA to segregate properly; reason not known
- ascertained by introducing phage DNA because yeast DNA could not be introduced because it might contain new or better elements or more of ARS, centromere, and telomere sequences