Wright 10 - Eukaryotic Gene Regulation and Chromatin Conformation

Question 1

What are the two components of chromatin?

Answer 1

DNA and protein

Question 2

When a bacterial cell is lysed, how does its genetic material come out?

Answer 2

A uniform chromatin structure of twisted loops come out. These contain supercoiled DNA, with the ends of the loops held in place by proteins.

Question 3

Name all the histones of eukaryotic chromatin. It may help to remember how many there are.

Answer 3

There are 5

• H1
• H2A
• H2B
• H3
• H4

Question 4

What are four common properties of histones?

Answer 4

1. Tightly bound to DNA, require a high salt concentration to extract the histones from DNA
2. High contents of basic (positively charged) amino acids interact with phosphate groups of DNA
3. Amino acid sequence of each of the histones is highly conserved from yeast to humans
4. Most abundant chromosomal proteins: 50% histones to 50% DNA, therefore, not regulators of specific genes but structural proteins in chromatin

Question 5

How are histones numbered?

Answer 5

According to their mobility on protein separation gels (eg. their molecular mass)

H1 is the heaviest, H4 is the lightest.

Question 6

What are the majority of non-histone chromosomal proteins (NHC proteins)?

Answer 6

High mobility group (HMG) proteins

Question 7

What are 5 properties of high mobility group non-histone chromosomal proteins?

Answer 7

1. Abundant chromosomal proteins, as such, not specific regulators of gene expression
2. Loosely bound to chromatin, extracted by low concentration of salts
3. High in basic (positively charged) and acidic (negatively charged) amino acids
4. More diverse in amino acid sequence of individual HMG proteins from one species to another
5. HMG proteins exhibit distinct tissue specific patterns of distribution

Question 8

What are non-high mobility group (HMG) non-histone proteins like?

Answer 8

They are in low abundance but are normally specific transcription factors such as steroid hormone receptor complexes

Question 9

What two experimental approaches defined the structure of chromatin during interphase?

Answer 9

1. Electron microscopy

2. Biochemical studies using nucleases, such as DNase I or micrococcal nuclease, to digest DNA in chromatin

Question 10

What are the two types of chromatin conformations observed in interphase nuclei?

Answer 10

• Beads-on-a-string. thin fibre (10 nm)

- Solenoid. thick fibre (30 nm)

Question 11

How is chromatin constitution assayed by biochemical methods?

Answer 11

1. Purified nuclei are lysed to release interphase chromatin
2. Chromatin were then subjected to digestion with nuclease (DNase I or micrococcal nuclease) for either short or long periods of time
3. The particles were then sedimented by centrifugation
4. Protein and DNA constituents of the sedimented material were purified and assayed by gel electrophoresis

Question 12

What is the result of nuclease digestion of eukaryotic chromatin for a short time?

Answer 12

Showed bands at integers of 200 base pairs. This was evidenc that DNA is organized into fundamental units of 200 bp in eukaryotic chromatin

Question 13

What did nuclease digestion of eukaryotic DNA for a long time show?

Answer 13

That DNA bands were split by integers of 150 bp, showing evidence that it is further organized into 150 bp

Question 14

What was concluded about short nuclease digestion yielding 200 bp strands and long digestion yielding 150 bp strands?

Answer 14

That 200 bp strand are organized by all five histones including H1. And that if allowed to digest for even longer then they are split into 150 bp fragments, including four histones but not H1.

Short digestion yielded single nucleosomes at 200 bp. Long digestion destroyed the linker DNA on H1 and released it from the core nucleosome (150 bp)

Question 15

What are two possible conformations of 30 nm fibres of chromatin?

Answer 15

• Solenoid one-start helix

- Zigzag two-start helix

Question 16

How is eukaryotic chromatin not free floating?

Answer 16

Chromatin loops are attached to an internal protein sub-structure called the nuclear matrix scaffold, or cage

Question 17

What is special about chicken erythrocytes?

Answer 17

They have nuclei, unlike most mammal’s, which do not.

Question 18

What is the consequence of having chromatin in the condensed 20nm conformation?

Answer 18

It is more resistant to nuclease digestion and transcriptionally inactive.

Transcriptionally active chromatin is in the extended 10 nm conformation and is more rapidly digested by nucleases.

Question 19

What controls the transition of chromatin from the condensed (30 nm fibre to the extended 10 nm fibre) forms, and vice versa?

Answer 19

The acetylation and deacetylation of nucleosomal histones. Histone acetyltransferase (HAT) adds acetyl groups to the histone tails. Histone deacetylase removes the acetyl groups.

Question 20

What does acetylation of histones do?

Answer 20

It weakens their interaction with DNA. The acetyl group also likely repels adjacent nucleosomes, thereby decondensing the chromatin and allowing access off some transcription factors to DNA.