LEcture 1: DNA-Chromosomes and Packaging

What organisms did eukaryotes evolve from?

Archaea endosymbiosed bacterial cells.

Shared features of eukaryotes and prokaryotes (4)

1. separation from the extracellular environment via a plasma membrane

2. proteins synthesized by ribosomes

3. ATP is used to store energy

4. Genetic material is encoded by DNA

How is DNA storage different between Euks and Pros

Stored in nucleus as long linear chromosomes in eukaryotes, more frequently stored as plasmids in bacteria (not in a nucleus)

How did eukaryotes arise (describe process)

1. Archael protrousions expanded around a bacterium (with its own DNA)

2. The protrusions closed around the bacterium

3. The endosymbiont escaped into the cytosol and new intracellular compartments began to form.

semiconservative replication

each resulting strand contains one new and one original strand

gene

segment of DNA that encodes a functional entity (protein, tRNA, rRNA)

genome

all the genetic informaiton in an organism

how many chromosomes do humans have

46 total chromosomes, 23 pairs of chromosomes

during what part of the cell cycle do chromosomes become compacted?

cell division

Appearrance of chromosomes in non-dividing cells

spread out into territories

karyotyping

genetic test involving arraging a person’s chromosomes to see whether they have a full set of chromosomes

How is it possible that DNA can be packaged?

DNA interactions with histomes

nucleosome

DNA wrapped around one histone complex

Chromatin

DNA plus associated proteins; i.e., nucleosomes interact with eachother to create chromatin fibers

What are some of the non histone proteins associated with DNA

regulatory proteins that control gene expression and higher order structure

euchromatin

DNa that is loosely packaged

Heterochromatin

DNA that is lightly packaged

Between heterochromatin and euchromatin, which is more accessible for expression?

Euchromatin

Can DNA around histones be accessed?

Yes! (becomes unwrapped temporarily)

What determines euchromatin v heterochromatin

higher order structures (no-matter whether euchromatin or heterochromatin, DNa is nearly always wrapped around histones)

How is the appearance of euchromatin different from heterochromatin when viewing a specific gene under fluorescence microscopy

the euchromatin gene is more extended and open while the heterochromatin gene is condensed and near the nuclear periphery

What does methylation marking on DNA indicate

Methylation of Cytosines (on CG sequences) marks DNA for silencing; methylation can designate regions of heterochromatin

DNA methyltransferase

adds methyl group

DNA demethylase

removes methyl group

Can methylation of DNA be maintained after DNA replication?

YES!

What are the names of the histone proteins that make up the nucleosome

H2A, H2B, H3, and H4

How is a histone assembled

two copies of each histone protein forms and octomer and there are unstructured tails that extend out of the nucleosome beyond the DNA.

What is the sigificance of histone tails?

thought to play an important role in associating nucleosomes to assemble chromatin fibers; different modificatios can affect the packing of DNA and how tight the histome holds onto the DNA.

What histone protein is a common target of modification?

H3

H3K9AC is associated with which chromatine type?

highly accessible, open chromatin

H3K9ac gene expression

on

H3K9me3 is associated with which chromatin type?

heterochromatin (either constitutive or facultative)

H3K9me3 gene expression

off

H3K27me3 associates with which type of chromatin

facultative heterochromatin

constitutive histone modification

permanent

facultative histone modification

temporary

H3K27me3 gene expression

OFF

histone writer

enzyme that creates a particular modification on one of the for nucleosomal histones

How is a histone writer recruited to a specific site on a chromosome, and what does it do once it is localized to that site?

One example of recruitment to a specific spot is by a transcription regulator protein; once there, the writer collaborates with a reader protein to spread its mark from nucleosome to nucleosome by means of the write-reader complex

What is necessary for the histone reader-writer complex to work?

the reader must recognize the same histone modification mark that the writer produces, and the binding mark of the reader should activate the writer

What enzyme/enzyme complex can undo the histone modifications caused by reader-writer complexes?

A reader-eraser complex

what limits the spread of heterochromatin (through the action of histone reader/writer complexes)

barrier sequence

What type of signal recruits a histone-modifying enzyme?

a repressive signal

What can modifications by the histoe reader-writer complex recruit

a DNA methylase that methylates the local DNA sequences (methyl groups added to the DNA are much more permanent than histone modifications)

Which causes more permanent effects? Methylation of the DNA sequence or methylation of a histone protein?

Methylation of a DNA sequence

barrier DNA sequence

prevets heterochromatin from spreading into areas that need to stay open

Mechanism by which barrier sequences block the spread of heterochromatin

1. recruit proteins that block the reader-writer complex

2. physically block the binding sites

3. acetylate histomes (H3K9ac) to designate that the region needs to stay open

How can DNA elements interact with other DNA elements that are multiple kilobases apart?

through the formation of chromatin loops

CTCF complex

sequece specific DNA-binding protein; forms a protein complex that stalls/stops the moving of a coheshin ring, and also holds the two ends of the DNA together

What class of proteins does CTCF belong to?

insulator proteins —> helps maintain the discrete domains of chromatin function

Cohesin

Protein that forms a small loop in the DNA and uses ATP to pull the loop of DNA longer

What happens when CTCF monomores interact at the base of each chromatin loop?

They bind to each other and stabilize the chromatin loop. → i.e., the string cannot go any further because the CTCF on the loop is stopping more DNA from being pulled through.

What method should you use to determine what sequences of DNA interact with eachother

HI-G

How does the HI-C assay work?

1. formaldehyde crosslinks everything

2. Restriction nucleases “trim the DNA dowm” → get rid of the long loops, leave sticky ends

3. Biotin marking (makes it easier of identify) —> soon both yerminal biotins witll be remoed

4. Ligation sticks DNA strands together if they are in close proximity to other DNA strands

5. isolate the biotin bound fragments, bind the DNA, run PCR to amplify

Durig HI-C, what are the only types of DNA sequences that are remaining at the end?

DNA product is obtained only if the proteins hold the two sequeces close to gether in the cell.

What are the potential outcomes of the HI-C expreiment?

1. filtered data: see which sequences are associated with your DNA region of interest by

2. unbiased: create an interaction map of how often you find different sequences interacting

In HI-C data, what does a strong diagonal line cell you

you get strong association of your DNA of interest with neighboring sequences

In HI-C data what does a strong signal far away form the diagonal indicate?

loops that are farther apart are interacting

TADs

topologically associated domains; any two DNA sequences within the domain are more likely to encounter eachother than with DNA sequences outside the domain

condensins

can form loops within loops to creasts very condensed chromosome

Do cohesins stick together loops formed by condensins or do condensins stick together loops formed by cohesins>

condensins stick together loops formed by cohesins

how does the nonsense mediated mRNA mechanism begin?

as an mRNA is being transported from the nucleus to the cytosol → As soon as the 5’ end emerges from the nuclear pore, the mRNA is met by a ribosome, which begins to translate it

In nonsense-mediated mRNA decay, what happens when the mRNA begins to be translated by a ribosome?

as translation proceeds, the JECs that are bound to the mRNA at each completed splice site are displaced by the moving ribosome

In the nonsense mediated mRNA decay path, what does a normal mRNA look like (that would not be sent for degradation)

the normal stop codon should lie within the last exon and the mRNA should be free of EJCs → in this case the mRNA passes inspection and it is released to the cytosol where it can be translated in earnest

In nonsense mediated mRNA decay, what happens if a ribosome reaches a stop codon early?

The EJCs would still be bound → the mRNA molecule would be rapidly degraded

What does the first round of translation allow the cell to do?

test the fitness of each mRNA as it exits the nucleus.

What interaction (specifically) triggers nonsense-mediated decay of mRNA

the UPF proteins that bind the EJC that encountered after the premature termination codon.