Gene Expression

Question 1

What are control elements?

Answer 1

They are sequences of DNA that allows the gene expression via the binding of specific transcription factors.

The proximal control elements are located close to the promoter, while the distal control elements may be far away from the gene, or even in an intron.

Question 2

What are enhancers?

Answer 2

Groups of distal control elements are called enhacers. which can be bound by transcription factors to enhance transcription for associated gene.

Question 3

What is an activator?

Answer 3

A protein (transcription factor) that binds to enhancers to stimulate transcription of a gene.

They can bind in or near a promoter.

Bound activators cause mediator proteins to interact with proteins at the promoter, stimulating transcription.

Question 4

Describe helix-turn-helix proteins.

Answer 4

• 2 alpha helices, between which there is a turn of short chain amino acids. Their helices are kept at a fixed angle as amino acids of these 2 interact via specific bonds.
• One helix enters the major groove of DNA and recognizes a specific sequence, thus it’s called a recognition helix.
• They are presented as dimers as they recognize DNA as dimers.
• The distance of DNA recognized is exactly 1 turn of helix.
• This way activators and oppressors both bind to DNA the same way, whether they are an activator or oppressor will be determined by other factors.

Question 5

Describe the leucine-zipper proteins.

Answer 5

• Shape resembles a zipper with 2 alpha helices joined at 1 point.
• Always bind to DNA as dimers.
• Uses hydrophobic amino side chains.
• Have a typical Y shaped form through which they get in contact with a major groove of DNA and recognize sequences of nucleotides.

Question 6

Describe beta-sheet recognition proteins.

Answer 6

• Uses 2 beta sheets to recognize DNA.
• They do still have alpha helices.
• Shown bound to a cofactor.

Question 7

Describe zinc finger proteins.

Answer 7

• Needs zinc to reach tertiary structure.
• Shape resembles a finger.
• Structure kept in its final working shape by zinc.

Question 8

Describe helix-loop-helix proteins.

Answer 8

• More similar to leucine zippers.
• Short alpha helices connected to a longer one via a loop.
• Makes different dimers - hetero and homodimers.

Question 9

What does dimerization of these factors achieve?

Answer 9

This increased the factor’s affinity and specificity for DNA.

Once one of the monomer has bound to DNA, this can make it easier for the 2nd one to bind. Therefore dimerization increases the ability to bind to DNA.

Question 10

What are operons?

Answer 10

A cluster of genes which are functionally related which undergo coordinated control by a single switch which can be on or off.

This switch is a part of DNA and is called an operator. This is a short stretch of nucleotides.

Operons include a structural, operator and regulatory gene.

Question 11

What is a repressor?

Answer 11

A protein that can inhibit gene expression by binding to silencers, they do this by inhibiting transcription.

In operons they can block gene expression by binding to the operator. This binding can block transcription of all genes in the operon.

This is produced by a specific regulatory gene outside the operon.

Question 12

What is a core repressor?

Answer 12

A molecule which coorporates with the repressor to inhibit operon expression (‘switch off’).

Question 13

How does tryptophan work as its own core repressor?

Answer 13

The tryptophan operon is composed of genes that transcribe tryptophan, therefore its on by default.

When tryptophan levels are low, the repressor is inactive as its unable to bind to the operator.

Although when tryptophan levels are too high, there will be enough present to bind to the tryptophan repressor, which will in turn bind to the operator to inhibit production.

Question 14

What is the function of the operator in operons?

Answer 14

Control the genes.

Question 15

Describes the 2 types of operons.

Answer 15

Repressible operon - this is usually on, binding of a repressor to the operator shuts off transcription. E.g. trp operon.

Inducible operon - this is usually off, an inducer is needed to inactivate the repressor and turn transcription on. E.g. lac operon.

Question 16

Describe operons in prokaryotic cells.

Answer 16

There are no operons in prokaryotic cells.

Question 17

Describe the lac operon.

Answer 17

An inducible operon that contains genes coding for enzymes involved in hydrolysis and metabolism of lactose.

The lac repressor is by defualt on and inhibits the expression of the lac operon.

However a molecule called the inducer can inactivate the lac repressor and turn it ‘on’.

Question 18

What are the different ways that activators can promote transcription?

Answer 18

• They can promote the binding of additional regulators.
• Recruit RNA polymerase to promoter.
• Release RNA polymerase from its pre-initiation condition to begin transcription.
• During elongation, the RNA polymerase can sometimes experience pauses. In this case the activator can release them from this.

Question 19

What’s the meaning to the statement that transcription activators work synergetically?

Answer 19

There is usually more than 1 transcription activators working on the promoter.

Question 20

How can histone modification help to regulate transcription?

Answer 20

Histone modifications lead to opening or closing of chromatin, which directly affects the access
of transcription factors to chromatin, thus affecting transcription.

Chromatin remodelling complexes - can remodel the chromatin to make the TATA box more or less accessible.

Recruitment of histone deacetylases - this opens up chromatin by acetylating histones. Histone deacetylase does the opposite, which makes chromatin more compact.

Recruitment of histone methyltransferase - the methyl groups of histones are bound by specific proteins, which makes chromatin compact and maintains in its silent condition.

Question 21

What are silencers?

Answer 21

A DNA sequence that binds repressors, a transcription factor that inhibits gene expression.

Question 22

How do repressors work?

Answer 22

Competitive binding - Competition for the same side in DNA between activator and oppressor.

Masking the activation surface - binding to a binding site and hiding the activation surface for the activator.

Direct interaction with the general transcription factors - can bind directly to the complex of RNA polymerase II and its cofactors.

Question 23

What signals regulate transcription factors?

Answer 23

Protein synthesis - the cell can synthesis transcription factors.

Ligand binding - the binding of a ligand (cofactor) makes an inactive transcription factor active.

Covalent modification - transcription factors need to be covalently modified to be active, phosphorylation usually employed.

Addition of a second subunit - needs addition of another subunit in order to make a complete and active transcription factor.

Unmasking - factor bound to an inhibitor that needs to be removed (usually by phosphorylation) in order for it to become active.

Stimulation of nuclear entry - needs to be inside the nucleus to work. Some are maintained by inhibitory proteins that prevent the factors from entering the nucleus, once they are removed, the transcription factor enters the nucleus and starts working.

Release from the membrane - stored in the membrane, can be cell membrane or of an internal organelle.

Question 24

What signals regulate transcription factors?

Answer 24

Protein synthesis - the cell can synthesis transcription factors.

Ligand binding - the binding of a ligand (cofactor) makes an inactive transcription factor active.

Covalent modification - transcription factors need to be covalently modified to be active, phosphorylation usually employed.

Addition of a second subunit - needs addition of another subunit in order to make a complete and active transcription factor.

Unmasking - factor bound to an inhibitor that needs to be removed (usually by phosphorylation) in order for it to become active.

Stimulation of nuclear entry - needs to be inside the nucleus to work. Some are maintained by inhibitory proteins that prevent the factors from entering the nucleus, once they are removed, the transcription factor enters the nucleus and starts working.

Release from the membrane - stored in the membrane, can be cell membrane or of an internal organelle. When the dignal from the extracellular environment arrives, this protein is activated by a proteolytic cut.

Question 25

Diffesrentiated cells will produce the same differentiated daughter cells, what does this tell us about its combination of transcription factors?

Answer 25

This shows that cells have a memory of the genes that were activated at the beginning of differentiation.

Question 26

How does transcription factors affect cell differentiation?

Answer 26

As the cell divides, they begin to differentiate from embryonic cells.

This involves transcription factors, as different combinations of transcription factors work to control the expression of different genes in different cells.

Consequently, each type of differentiated cell contains a specific combination of transcription factors that only express the genes needed by the cell to perform its function.

Question 27

How did Yamanaka and Gurdon turn fibroblasts to IPS (Induced Plueipotent Stem) cells?

Answer 27

They introduced 4 fundamental transcription factors to the fibroblast nucleus, then allowed the cell to divide in culture.

This created the IPS cells, which can then be induced to differentiate in culture.

Question 28

What are the 3 transcription factors that regulates a huge number of genes?

Answer 28

Klf4, Oct4, Sox2.

Question 29

How does the glucocorticoid hormone induce or inhibit gene expression?

Answer 29

The glucocorticoid hormone can pass through the PM as its a lipid. It binds with the glucocorticoid receptor present in the cytoplasm to form a complex, which then travels to the nucleus.

After reaching DNA, they bind to a regulatory DNA sequence and either induce or inhibit gene expression.

Question 30

How does differentiated cells maintain their memory and identity?

Answer 30

A transient signal arrives, causing an undifferentiated cell to produce specific transcription factors that causes it to become a precursor cell.

The transcription factor will also induce its own subsequent transcription and production.

The daughter cells from this precursor cell will therefore also have this transcription factor, continiously transcribing and producing itself.

In the subsequent generations, the memory of this factor will be maintained and the cell will remain differentiated, even if the initial signal is no longer there.

Question 31

Describe how DNA modifications can be inherited in epigenetics inheritance.

Answer 31

After DNA replication, an enzyme called maintenance methylase remembers where the previous strand was originally methylated and will methylate these bases.

Question 32

How does the methylase enzyme spread methylation along DNA?

Answer 32

DNA methylase uses code ‘reader’ proteins that spread along the DNA, to add methyl groups that also spreads.

DNA methyl binding proteins recognised the methyl groups in DNA and bind to them.

Question 33

What are CG islands?

Answer 33

Very long concentrated stretches of CG dimers, usually in the beginning of the promoter.

Regions that are very enriched in CG dimers are called CpG islands.

Cytosomes here are usually non-methylated.

Question 34

How does accidental deamination lead to loss of CPG islands?

Answer 34

Usually, if an accidental deamination of a methylated C occurs, a U would result. This is easily recognised as incorrect in DNA and is replaced by DNA repair systems.

Although if a non-methylated C is accidently deaminated, a T would result instead. This is not as easily recognised and therefore not repaired.

During evolution, as non-methylated Cs tend to mutate into Ts, this leads to a global loss of CgP dinucleotides.

Question 35

Why do CpG islands remain non-methylated?

Answer 35

This is important for the function of housekeeping genes.

Question 36

How can epigenetic inheritance be inherited from an organism to an offspring?

Answer 36

In mammals, the expression of genes sometimes depend on derivation from either maternal or paternal genes.

In the case of Igf2, only the paternal copy is expressed. Therefore if this is mutated in the paternal gene, then mouse offsprings are dwarf.

The maternal copy is supressed.

Question 37

Why is the igf2 gene imprinted in male chromosomes?

Answer 37

In the maternally inherited chromosome, there is an insulator element between the igf2 gene and its regulatory sequence (enhancer). The cofactor CTCF binds to the insulator element to separate igf2 from its regulatory sequence.

Consequently, transcriptuion facors can’t bind to this enhancer and thus the igf2 gene is not expressed.

While in the paternally inherited chromosome, the insulator element is methylated. This makes it invisble to CTCF, thus it can’t bind.

Therefore the enhancer can induce igf2 gene expression.

Question 38

Why is the kcnq1 gene imprinted in female chromosomes?

Answer 38

In the maternal chromosome, a methylation site is present and does not interfere with gene expression.

While in the paternal chromosome, this methylation site is absent. Consequently this uncovers a hidden promoter that transcribes in the opposite diection to the gene sequence in maternal chromosome.

This results in a non-coding incRNA. Histone modifying enzymes are recruited by RNA to repress the chromatin, resulting in this gene sequence not being expressed.

Question 39

What is a Barr body?

Answer 39

Condensed, inactive X chromosome in the female somatic cell.

Question 40

How are Barr bodies formed?

Answer 40

As females have 2 X chromosmes, one of them will randomly become very condensed when the embryo is a few hundred cells.

This is called X inactivation, and can happen to either the paternal or maternal X chromosome with a 50% chance for each.

The daughter cells of the somatic cell will have a memory of this condensation. Consequently all cells would express only the X chromosome that did not inactivate.

Question 41

How is the xist gene expressed before chromosome inactivation?

Answer 41

In a completely random way, one of the 2 X chromosomes will start expressing a gene called xist, which produces the xist RNA (non-coding).

This RNA can spread ‘handover-hand’ along the chromosome from where its produced. It can bind to histone modifying protein and contibue spreading at the same time.

The histone modifying proteins are repressive, therefore the chromosome that expresses xist will be inactivated.

Question 42

What are epigenetic mechanisms that act in cis? Give examples.

Answer 42

Affects only 1 copy of chromosome.

Examples are DNA methylation and histone modification.

Question 43

What are epigenetic mechanisms that act in trans? Give examples.

Answer 43

Affects both chromosomal copies.

Examples are the positive feedback loop by transcription regulators, and protein aggregation state.

Question 44

What s transcription attenuation?

Answer 44

The premature termination of some RNA molecules.

Question 45

How does transcription attenuation work in HIV infection?

Answer 45

When HIV infects cells, it produces a copy of its genome called proviral DNA. This will intergrate into the host’s genome.

After undergoing transcription, the viral RNA is not completely translated and prematurely terminated by RNA polymerase.

After some replication cycles when conditions become optimal for HIV growth, the protein tat (virally encoded) starts binding to tthe nascent viral RNA. This works as a protector for the RNA of the virus, which is now completely transcribed and translated.

Question 46

How does riboswitches control genes? Use purine as an example.

Answer 46

If there is a low number of purine, the gene for its synthesis is on and RNA polymerase is actively transcribing RNA. This RNA has a series of nucleotides, presenting a complex secondary structure.

Although if purine levels are high enough, it will interact with this secondary structure to expose a region that terminates the RNA polymerase.

As a result transcription is incomplete and purine synthesis stops.

Question 47

What are the different possible results of alternative splicing?

Answer 47

Exon skipping - 1 exon can be skipped and spliced away with the 2 introns.

Intron retention - the intron is included in the process.

Alternative 5’ splice site

Alternative 3’ splice site

Mutually exclusive exons - the splicing machinery will decide which exons to maintain or spliced away.

Question 48

How can splicing be controlled in negative ways?

Answer 48

When the repressor is present, it binds to the splicing silencer and represses the splicing of the intron in that region.

Question 49

How can splicing be controlled in positive ways?

Answer 49

Activators recognise and bind to splicing enhancers, which induces splicing of the introns in that region.

Question 50

How can the choice of polyadenylation sites affect the types of proteins synthesised? Use B cell antibodies as examples.

Answer 50

Resting B cell - B cell with membranbe bound antibodies due to low CstF levels.

There is a weak and strong section in a gene. The weak section is spliced out, and CstF binds to the 3’ end of mRNA. This will produce the longer type of this mRNA and the protein produced will be antibodies that bind B cells.

Activated B cell - B cell secrets antibodies, high CstF levels.

A high amount of CstF is produces, therefore allowing the weak section of the gene to be recognised and cleaved. This results in a shorter mRNA that encode for antibodies that will not attach to B cell, but in secreted form.

Question 51

What is RNA editing?

Answer 51

The sequence of a RNA can be changed by specific editing enzymes.

Question 52

How does ADAR work in RNA editing?

Answer 52

ADAR (Adenosine Deaminases Acting on RNA) in mammals - work by cutting amine group from adenosine, producing an intermediate called inosine. This is read by the cells as guanosine.

This results in a mutation from A to G.

Question 53

How does Rev regulate nuclear transport in HIV RNA?

Answer 53

In early HIV synthesis, 2 sections of mRNAs are transcribed. One of them is unspliced, containing the RRE region. This results in them being retained in the nucleus and degraded.

The other section is fully spliced and can exit the nucleus. From there they synthesise Rev and other early viral proteins. Rev is able to move back into the nucleus.

During late HIV synthesis, Rev moves into the nucleus and binds to the RRE region on the unspliced mRNA. Consequently the mRNA is able to exit the nucleus and synthesise all viral proteins.

Question 54

What are 3’ UTRs?

Answer 54

3’UTRs (Untranslated Region) are downstream of stop codons in RNAs so are not translated.

Although they are needed to define the post-translational fate of RNAs.

Question 55

What are the different ways that UTRs control translation?

Answer 55

A translation repressor binds to a secondary structure that doesn’t code for proteins, resulting in the ribosome not being able to bind to the start codon.

Affected by external conditions - temperature in this case. The mRNA is in itself sensitive to temperature changes. When it’s low, this structure is tightly closed so the ribosome is not able to approach and start translation.

Presence of small molecules can change the structure of UTR which hides the initiation codon.

Antisense RNA that is complementary to mRNA, binds with mRNA and hides initiation codon.

Question 56

How does the phosphorylation of an initiation factor regulate protein synthesis?

Answer 56

eIF2B changes the GDP attached to elF2 to GTP.

After elF2 is phosphorylated by protein kinase, it binds to elF2B as an inactive complex. In the absence of elF2B, the remaining elF2 stays inactive.

As a result, protein synthesis decreases.

Question 57

What are Internal Ribosomal Entry Sites (IREs)?

Answer 57

A specialised type of RNA sequence that doesn’t need 5’ capping and the eIF4E protein.

Question 58

Provide some examples of when IREs are used.

Answer 58

• By viruses to take control of the cellular translation machinery.
• Some specific mRNAs during M phase.
• Some specific mRNA during apoptosis.

Question 59

Hoe can IREs control translation?

Answer 59

IREs do not depend on mRNA cap and its binding factor eIF4E, therefore the proteins it synthesises can be selectively increased in some circumstances.

Question 60

What determines stability of mRNA?

Answer 60

mRNA experiences continious shortening of their poly-A tails. At a certain point, their tail will shorten enough for them to be degraded.

Question 61

What does deadenylase do?

Answer 61

An enzyme that degrades the poly-A tail of mRNA whilst its working in translation. These 2 machines ‘compete’.

Question 62

How is iron managed in cells?

Answer 62

Iron starvation -

Question 63

What are P bodies and stress granules?

Answer 63

Stress granules are formation in cytoplasm of eukaryotic cells induced by different types of stress, such as heat and starvation.

P bodies are very similar and present almost always in many types of eukaryotic cells.

It’s been found that mRNAs have been stored here whilst waiting for their fate, i.e. to be degraded or start another round of transcription.

Question 64

How can some non-coding RNAs regulate gene expression?

Answer 64

The entrance of a double stranded RNA in a cell results in its processing into shorter and non double stranded, called interfering RNA.

This has a high leve of complementarity to a target RNA, and binds to it. This can result in:

Cleavage of target RNA - name of the interfering RNA is siRNAs (short interfering RNAs).

Translational repression and eventual destruction of target RNA - miRNAs (microRNAs).

Formation of heterochromatin on DNA from which the target RNA is being transcribed - rasiRNA (repeat associated small interfering RNA).

Question 65

How does microRNAs work?

Answer 65

They work in a way that is endogenous to cells.

miRNAs are loaded onto specific protein complexes. Depending on how much they hybridize to target RNA, they can either degrade mRNA; or cause translational repression and degradation.

Question 66

How does siRNAs work?

Answer 66

An invader double-stranded RNA (viral RNAs) is processed into siRNAs.

They can work by being loaded by different proteins, and induce a heterochromatic state in chromatin.

Question 67

Describe how RNA interference is also used as a cell defense mechanism.

Answer 67

In some organisms, e.g. worms and plants, RNAi can spread between tissues by movement between cells.

This can lead to the whole organism being resistant to an infection.

Question 68

How does bacteria protect themselves from viral injection? (Experimental methods).

Answer 68

The bacterial cell is infected by the virus, which injects its genome into bacterial DNA. The viral DNA is cleaved into short pieces and integrated into bacteria genomic DNA, and this happens each time a new cell is invaded. This starts a cell memory of the virus DNA sequences. This is called CRISPR locus.

The CRISPR loci is transcribed into RNA processes and each of the short pieces is bound to CAS proteins.

When the same type of virus infects the cell, it will inject its DNA again inside. However this will now be recognized by one of the particles containing a short piece of the same viral DNA. The small crRNA is as complex as CAS seeks out and destroys viral sequences.

Question 69

What functions fo long non-coding RNAs have in the cell?

Answer 69

They can act in cis - controls transcription of genes on the same chromosome they were transcribed.

In trans - can move from their original gene and control transcription of genes on another chromosome.