19 Gene Expression in Eukaryotes

Question 0

How many base pairs of DNA are in a typical human cell?

Answer 0

6billion

Question 1

Differential gene expression

Answer 1

Is responsible for creating different cell types, arranging them into tissues, and coordinating their activity to form the multicellular society we call an individual

Question 2

How long would a fully stretched DNA molecule be in humans?

Answer 2

2meters (6.5feet)

Question 3

What is the nucleus diameter?

Answer 3

5 micrometers

Question 4

Chromatin layers of organization

Answer 4

1. DNA wrapped around 8 histones to form nucleosomes
2. Nucleosomes are packed into 30nm fibre
3. 30nm fibre attached to protein scaffold
4. entire assembly folded into a highly condensed structure observed during cell division

Question 5

H1 protein function

Answer 5

Seals DNA to each nucleosome

Question 6

Linker DNA

Answer 6

Short chain of DNA that links nucleosome to another nucleosome.

Question 7

Central idea is that the chromatin must

Answer 7

Decondense to expose the promoter so RNA polymerase can bind to it

Question 8

Function of DNase?

Answer 8

Enzymes that cut DNA

• cleave DNA at random locations
• cannot cut efficiently if tightly wrapped with proteins

Question 9

Harold Weintraub and Mark Groudine

Answer 9

DNA of actively transcribed genes is in an open configuration

• compared chromatin structures of two genes, β-globin and ovalbumin, in chicken blood cells.
• β-globin is protein in haemoglobin found in red blood cells, ovalbumin is a protein found in egg white.
• in blood cells, β-globin is transcribed at high levels, but ovalbumin is not at all.
• then treated cells with DNase
• found that the β-globin gene was cut more readily
• CONCLUSION: chromatin of the actively transcribed β-globin was decondensed, conversely, the chromatin of ovalbumin was condensed.

Question 10

Histone gene repression

Answer 10

The absence of histone-DNA interactions promotes transcription
The presence of normal histone-DNA interactions prevents it

Data suggest that in their normal, or default state, eukaryotic genes are turned off.

Different form of negative control

Question 11

DNA methylation

Answer 11

1. Enzyme DNA methyltransferase adds methyl group (-CH3) to cytosine residues in DNA.
2. In mammals, the sequence recognized is a C next to a G in one strand of DNA. Abbreviated CpG
3. Methylated CpG sequences are recognized by proteins the trigger chromatin condensation.
4. Actively transcribed genes usually have low levels of methylated CpG

Question 12

Histone modification

Answer 12

Large set of enzymes adds a variety of chemical groups to specific amino acids of histone proteins.

• phosphate groups
• methyl groups
• short polypeptide chains
• acetyl groups (-COCH3)

Question 13

Histone code hypothesis

Answer 13

Postulates that particular combinations of histone modification set the stage of chromatin condensation for a particular gene

Important role in regulating transcription

Question 14

Histone acetyltransferase

Answer 14

(HATs) Adds acetyl groups to the positively charged lysine residue in histones

Question 15

Histone deacetylases

Answer 15

(HDACs) remove acetyl groups

Question 16

Acetylation

Answer 16

The adding of acetyl groups to histones

-usually results in decondensed chromatin, state associated with active transcription

Question 17

How can acetylation of histone promote decondensation?

Answer 17

When HATs add acetyl groups, the acetyl group neutralizes the positively charges lysine residue loosens close association of nucleosomes with the negatively charged DNA.

The addition of the acetyl group also creates a binding site for other proteins to help open the chromatin.

Question 18

Chromatin-Remodeling complexes

Answer 18

1. Enzymes form macromolecule machines called chromatin-remodeling complexes.
2. These machines use ATP to reshape chromatin
3. causes nucleosomes to slide along the DNA
4. in some cases, knocks out the histone completely off the DNA
5. Opening up stretches of chromatin and allow gene transcription

Question 19

Gene associated with diabetes?

Answer 19

Hnf4a gene

Question 20

Promotor

Answer 20

Is a site in DNA where RNA polymerase binds to initiate transcription

Question 21

Eukaryote promotors

Answer 21

Are more complex than bacterial promotors

Containing two to three conserved sequences that serve as binding sites

Question 22

Most extensively studied promotor

Answer 22

TATA box

Question 23

After chromosome remodeling, what binds to the TATA box?

Answer 23

TATA-binding protein (TBP)

Does not guarantee that the gene will be transcribed

Question 24

Who discovered regulatory sequence?

Answer 24

Yasuji Oshima

Question 25

How do yeast cells control the metabolism of the sugar galactose?

Answer 25

When galactose is absent, yeast cells produce very little enzymes required to metabolize it. When present these enzymes increase by a factor of 1000 They found mutant that could not produce any of the five enzymes 1. The five genes are regulated together, even though they are not on the same chromosome 2. Normal cells have an activator protein that exerts positive control over the five genes 3. The mutant cells have a mutation that completely disables the activator protein.

Question 26

Promoter-proximal elements

Answer 26

Regulatory sequences located close to the promoter and bind regulatory protein Unlike the promoter itself, they have sequences unique to specific sets of genes. This way they express certain genes wanted but not others

Question 27

Enhancers

Answer 27

Regulatory sequences that are far from the promotor and activate transcription - positive control - can be more than 100,000bases away - can be located on introns or untranscribed sequences - can be on either 5' or 3' side of gene - many different types exist - usually have binding sites for more than one protein - can work even if their 5'-3' orientation is flipped - can work if they are moved to a new location

Question 28

Transcriptional activators

Answer 28

Regulatory proteins that bind to enhancers beginning transcription

Question 29

Silencers

Answer 29

Regulatory sequences that inhibit transcription.

Question 30

Repressors

Answer 30

Regulatory protein that bind to silencers shutting down transcription

Question 31

Collective term for promotor-proximal elements, repressors and transcriptional activators

Answer 31

Regulatory transcription factors | Transcription factors

Question 32

What causes cell differentiation

Answer 32

Different cell types express different genes depending on the transcription factors. Multicellular organisms can have transcriptional factors produced by signals from other cells (early in embryonic development)

Question 33

How do transcriptional factors recognize the specific DNA sequences?

Answer 33

- DNA sequences are partially exposed in the major and minor grooves. - Depending on the base pairing, there are different shapes and composition of atoms. - Transcriptional factors can recognize these different shapes and composition.

Question 34

Basal transcription factors

Answer 34

These are proteins that interact with the promoter and are not restricted to particular genes or cell types. Are necessary for transcription to occur but do not provide much in the way of regulation Eg. TATA-binding protein (TBP)

Question 35

Large complex proteins that act as a bridge between regulatory transcription factors, basal transcription factors and RNA polymerase II

Answer 35

Mediator

Question 36

Steps of initiation of transcription in eukaryotes

Answer 36

1. Transcriptional activators bind to DNA and recruit chromatin-remodeling complexes and histone acetlytransferases (HATs) 2. The chromatin-remodeling complexes and HATs open a swath of chromatin that includes promoter, promoter-proximal elements and enhancers. 3. Other transcriptional activators bind to the newly exposed enhancers and promoter-proximal elements. Basal transcription factors bind to the promoter and recruit RNA polymerase II 4. Mediator connects the transcriptional activators and basal transcription factors that are bound to DNA. This step is made possible through DNA looping. RNA polymerase II can now begin transcription

Question 37

RNA interference

Answer 37

Occurs when a tiny, single-stranded RNA held by a protein complex binds to a complementary sequence in an mRNA. This event unleashes either the destruction of the mRNA or a block in the mRNA's translation.

Question 38

RNA interference steps

Answer 38

1. RNA interference beings when RNA polymerase transcribes genes that code for RNAs that double back on themselves to form a hairpin. 2. Some of the RNA is trimmed by enzymes in the nucleus; then the double stranded hairpin that remains is exported to the cytoplasm. 3. In the cytoplasm, another enzyme cuts out the hairpin loop to form double-stranded RNA molecules that are only about 22nucleotide long 4. One of the strands from the short RNA is taken up by a group of proteins called RNA-induced silencing complex (RISC). The RNA hold by a RISC is a microRNA (miRNA) 5. Once it is part of a RISC, the miRNA binds to its complementary sequence in a target mRNA 6. If the match between a miRNA and an mRNA is perfect, an enzyme, in the RISC, destroys the mRNA by cutting it in two 7. If th match is not perfect the mRNA is not destroyed but translation is inhibited.

Question 39

Proteasome

Answer 39

Recognizes protein with ubiquitin tag and destroy it

Question 40

Compare DNA packaging between bacteria and eukaryotes

Answer 40

Eukaryote DNA must decondense for basal and regulatory transcription factors to gain access to genes and for RNA polymerase to initiate transcription. The tight packaging of eukaryote DNA means that the default stage of transcription is off In contrast, bacteria lack histone histones and have freely accessible promoters, so may be on in default stage. Therefore, chromatin structure of eukaryotes provide a mechanism of negative control not found in bacteria.

Question 41

Compare complexity of transcription between bacteria and eukaryotes

Answer 41

Much more complex in eukaryotes The sheer number of proteins involved in regulation transcription dwarves that in bacteria, as does the complexity of their interactions

Question 42

Compare coordinated transcription between bacteria and eukaryotes

Answer 42

In bacteria, genes that take part in the same cellular response are often organized into operons and are transcribed together by the same promotor In eukaryotes, like bacteria many genes can be expressed together, however, use regulons that have physically scattered genes expressed together (may not even be in the same chromosome) and regular transcription factors trigger the transcription of genes with the same DNA regulatory sequence

Question 43

Compare the reliance on post-transcriptional control

Answer 43

Eukaryotes make greater use of post-translational control, such as alternative splicing. Alternative splicing allows eukaryotes to produce many different proteins from the same gene. They also make microRNAs and regulate the stability of mRNA. These post transcriptional controls are seldom seen in bacteria

Question 44

What two classes of genes that when mutated may lead to cancer?

Answer 44

1. Genes that stop or slow the cell cycle | 2. Genes the trigger cell growth and division by initiating specific phases in the cell cycle

Question 45

What gene is most linked human cancers?

Answer 45

p53 - codes for regulatory transcription factors - halting cell cycle when DNA is damaged When a mutant form of p53 is formed enhancers cannot bind. DNA damage cannot arrest the cell cycle. The cell cannot kill itself and damage DNA is replicated

Question 46

Results that support the model function of p53

Answer 46

1. p53 activates many different genes, including genes for cell cycle regulation, DNA repair and apoptosis 2. X-ray crystallography studies show that p53 binds directly to DNA regulatory sequences of the genes it controls 3. Virtually all p53 mutations associated with cancer are located in the proteins DNA-binding region and alter amino acids that interfere with p53's ability to bind to regulatory DNA sequences