Transcription II

Question 1

What are genetic switches?

Answer 1

They are composed of gene regulatory proteins and the specific DNA sequences that these proteins recognize.

Question 2

In bacteria, the expression of many genes is regulated broadly by what?

Answer 2

The available food in the environment.

Question 3

What are the three types of proteins that regulate transcription?

Answer 3

• specificity factors: alter the specificity of RNA pol for a promotor or set of promotors (e.g. sigma factor in bacteria)
• repressors: impede access of RNA pol (called operators in bacteria)
• activators: enhance polymerase promotor interactions.

Question 4

Describe the prokaryotic lac operon

Answer 4

A cluster of genes coordinately regulated and transcribed together that regulate conversion of beta galactosidase into galactose and glucose.

Question 5

Describe two modes of negative regulation.

Answer 5

• Activator dissociated from activator binding site by molecular signal
• activation of repressor by molecular signal causing binding to operator.

Question 6

Describe two modes of positive transcription regulation.

Answer 6

• Molecular signal causes dissociation of repressor from operator.
• Molecular signal causes binding of activator protein to activator binding region, initiating transcription.

Question 7

What is the function of beta galactosidase?

Answer 7

It cleaves lactose to form glucose and galactose.

Question 8

How is the lac operon negatively regulated?

Answer 8

Binding of repressor protein to lac operator downstream of promotor which physically blocks RNA polymerase.

Question 9

Describe regulation of the lac operon when both lactose and glucose are present in the cell.

Answer 9

• lactose binds to repressor protein, changing its conformation so it cannot bind the operator (lac operon is no longer repressed)
• lac operon still not transcribed, however, because CAP protein is not active (only activated in the absence of glucose)

Question 10

Describe regulation of the lac operon when lactose but not glucose is present in the cell.

Answer 10

Lac operon is turned on:

• lactose binds and turns off repressor from binding operator
• in absence of glucose, adenylate cyclase is active and makes cAMP which binds and activates trans-acting CAP protein
• CAP binds enhancer region and helps RNA polymerase initiate txn at promotor site

Question 11

What is the difference between lac operon and trp operon?

Answer 11

The lac operon is controlled like a switch, but the trp operon is controlled like a dimmer.

Question 12

How is the trp operon coordinately controlled?

Answer 12

All five genes are transcribed from a single promotor as one long mRNA.

Question 13

Describe regulation of trp operon under low trp conditions.

Answer 13

The repressor is inactive and RNA pol transcribed all 5 genes of the operon.

Question 14

Describe regulation of trp operon under high trp conditions.

Answer 14

The repressor is activated by trp and binds the operator.

Question 15

What type of transcription factor is the trp operon repressor?

Answer 15

It has a HTH DNA-binding domain.

Question 16

Describe the elements of the trp operon.

Answer 16

• promotor
• 3 operators (trp-sensing)
• charged tRNA(trp) sensing transcription termination
• attenuator
• 5 structural genes

Question 17

Describe how the trp operon acts as an attenuator to dial-down expression.

Answer 17

It relies on the secondary structure of the trp operon mRNA.

• Attenutation relies on regions 1 and 2 regions 3 and 4 of the trp leader sequence to base pair and form hairpins (the latter being a termination sequence). This is the normal, stable structure.
• When trp is scarce, the ribosome stalls on region 1 at codons coding for trp, as there is no trp to allow translation, allowing regions 2 and 3 to form hairpin. This prevents the hairpin between regions 3 and 4 (terminator sequence), so RNA polymerase can continue transcribing without being terminated.
• When trp is present, the ribosome continues along the operon mRNA and stopping at stop codon, blocking region 2, allowing the terminator sequence structure between regions 3 and 4 to form. Thus, transcription is terminated at the second hairpin.

Question 18

What are the major differences between prok and euk gene regulation?

Answer 18

• prok cells control gene expression with one or very few proteins, but euk use hundreds of different regulatory proteins.
• the mediator in euk during transcription serves as an intermediary between gene regulatory proteins and RNA polymerase
• euk DNA is packaged in chromatin
• euk txn required GTF’s, allowing for multiple steps to speed up or slow down txn
• euk cells lack operons (are monocistronic) and regulate each gene individually

Question 19

Where on DNA do euk activators and repressors bind?

Answer 19

Either upstream or downstream of promotor, oftentimes far distances away.

Question 20

How do co-activators in euk interact with DNA?

Answer 20

They bind the transactivation domains of activators.

Question 21

Give an example of a euk co-activator.

Answer 21

Histone acetylase

Question 22

GIve an example of a euk co-repressor.

Answer 22

HDAC.

Question 23

Define the histone code.

Answer 23

The well-defined regulation of chromatin.

Question 24

Which residues of histones are acetylated by HAT?

Answer 24

K9 of histone 3 and K8 of H4.

Question 25

Which histone residues are phosphorylated by histone kinase, and what is the effect?

Answer 25

Ser10 of H3 is phosphorylated by histone kinase, signaling HAT to acetylate K14 of H3. This causes the recruitment of TFIID.

Question 26

How can we measure the activity of a co-activator or co-repressor protein?

Answer 26

We can fuse the promotor where the activator and co-activator bind with the promotor and coding region of luciferase or another reporter, and measure the activity.

Question 27

What are the mechanisms of txn repression in euk?

Answer 27

- Compeititve binding: Repressor binds where the activator would bind. - Masking the activation surface: Repressor transactivation site will bind that of the activator, so other factors can't bind it, - Direct interaction with the GTF's: Repressor binds TFIID so that activator cannot bind it - Recruitment of chromatin remodeling factors: histones near the TATA box and promotor might be deacetylated or methylated.

Question 28

How can the activity of a TF be restricted to a certain time or cell type?

Answer 28

1. factor is generally not present but synthesized only where it is needed (e.g. MyoD) 2. the factor is present but must be modified to be active (i.e. B-catenin/armadillo needs to be phosphorylated) 3. ligand is required for activity (nuclear/steroid hormone receptor) 4. localization of factor to inactive compartment of cell and required cleavage for activity (sterol response factors) 5. Factor may be bound by an inhibitory factor in the cell (NF-kB) 6. Dimeric factor may have multiple partners, some making it active and others not (bHLH and bZip) 7. Unmasking of activation domain by phosphorylation of inhibitor protein (E2F's involved in cell cycle regulation)

Question 29

What is the role of the nuclear/steroid receptor?

Answer 29

- To orchestrate embryogenesis - maintain homeostasis

Question 30

Describe cell surface nuclear/steroid receptors.

Answer 30

- membrane anchored and ligands change the activity of a nuclear transcription factor - signal factors (ligands) are hydrophillic and cant cross the cell membrane, hence the receptor being in the plasma membrane.

Question 31

Describe intracellular nuclear/steroid receptors.

Answer 31

- they transduce their signals directly - steroid hormones, thyroid hormones, retinoids and vitamin D are all small molecules that diffuse through the cell membrane and bind inside the cell

Question 32

What are the three classes of nuclear receptors?

Answer 32

1. steroid hormone receptors (bind inside cytoplasm and move to nucleus) 2. nuclear hormone receptors (bind inside nucleus) 3. orphan receptors (anything else)

Question 33

Describe the genetic makeup of the nuclear/steroid receptor gene.

Answer 33

- zinc finger DNA binding domain w/ dimerization motif - ligand binding domain w/ dimerization motif - transactivation domain for HAT interaction - very high affinity and selectivity

Question 34

Describe 3 hormone signaling pathways.

Answer 34

1. endocrine: on distant cells 2. paracrine: on neighboring cells 3. autocrine: on self

Question 35

Which biological processes are hormone signaling pathways involved in?

Answer 35

- reproduction - metabolic rate - stress - calcium homeostasis - vitamins \*all have homology in DBD

Question 36

Describe the N-terminal A/B domains in hormone receptor genes.

Answer 36

They vary in length, have differing sequences, and contain oone or more activation domains.

Question 37

Describe the interactions of steroid hormone receptors.

Answer 37

- they bind DNA as homodimers - synthesized in inactive forms associated with HSPs - ligands are steroid hormones -

Question 38

Describe cys-cys fingers (coordinated with Zn) in steroid receptors.

Answer 38

- first finger controls DNA binding - first finger contains P-box, a region that determines target specificity - both fingers bind the same half sites - second finger is responsible for ligand dimerization - spacing between the fingers can vary

Question 39

What is the role of spacing between Zn fingers in steroid receptors?

Answer 39

Spacing determines specificity, determined by D-box.

Question 40

How do E2F transcription factors regulate the cell cycle?

Answer 40

They form heterodimers with the DP members and activate genes responsible for cell cycle control, initiation of replication, and DNA synthesis.

Question 41

What point of the cell cycle is regulated by E2F transcription factor, and what factors influence its activity?

Answer 41

It regulates the restriction point, or start point transition into S phase. It is influenced by: - growth factors - nutrients - cell size - DNA damage

Question 42

How is transcription factor E2F regulated?

Answer 42

It is repressed by pocket proteins like Rb by masking its transcriptional activation domain. In the presence of growth factors, Cdk/cyclin heterodimer phosphorylates Rb (through Ras pathway), causing its release from E2F and the cell cycle to start.

Question 43

What type of gene is TF p53?

Answer 43

A tumor supressor gene, mutated in 50% of cancers.

Question 44

What is the role of transcription factor p53?

Answer 44

It prevents the cell from completing the cell cycle if DNA is not properly replicated in S phase.

Question 45

Which residue of p53 is most commonly mutated in cancers?

Answer 45

R248, causing loss of contact with DNA and p53's products are not expressed.

Question 46

How does txn factor p53 halt the cell cycle?

Answer 46

- p53 binds and induces txn of the cyclin-dependent kinase inhibitor p21/CiP1, blocking cdk2 and arresting cell cycle - when cdk2 is blocked, phosphorylation of Rb cannot occur

Question 47

During which phases of the cell cycle does p53 work?

Answer 47

Both G1 and G2 phases.

Question 48

Under what conditions do p53 expression levels increase in the cell?

Answer 48

Under high events of DNA damage.

Question 49

Which genes regulate p53 expression?

Answer 49

p53 is activated by phosphorylaion in the ATM pathway. It is held in an inactive state by mdm2. When p53 is highly expressed, mdm2 becomes highly expressed to bind and lower p53 activity,

Question 50

How many transcription factors regulate puripotent stem cells?

Answer 50

Four: oct4, sox2, klf4, c-myc