transcriptional circuits

Question 1

define transcriptome

Answer 1

The segment that is transcribed

Question 2

what are the 4 levels of gene transcription

Answer 2

• abundant transcript ( gene gives rise to many mRNA transcripts). If one in every cell this is called a housekeeping gene ( e.g glycolytic enzymes ) - genes that are required for the functioning of all cell types
• no transcript - not expressed or expressed in low levels.
• • Rare transcript -may not be expressed in one cell type, however it can be highly expressed in another tissue

• Rare or no transcript - stimulus - abundant transcript.

Question 3

what is an inducible gene

Answer 3

gene that becomes abundantly expressed in response to a stimulus such as hormones or viruses.

Question 4

what is a promotor

Answer 4

The sequence immediately 5’ to the region to be transcribed is called a “promoter”
•Promoters recruit RNA polymerase to a DNA template

Question 5

what is an enhancer

Answer 5

• Sequences of DNA that are not immediately adjacent to where transcription starts that act to enhance the recruitment of RNA polymerase to a promoter
• Enhancers can reside 5’ or 3’ to a transcription unit, and can even be located within an intron
• Like promoters, enhancers contain DNA sequences that are very strong binding sites for specificity factors or “transcription factors”

Question 6

what happens when RNA is recruited.

Answer 6

• Neither prokaryotic nor eukaryotic RNA polymerases make stable contacts with DNA – they slide along the duplex without being able to efficiently recognise promoters
• Once stably recruited to DNA, RNA polymerase is able to convert the DNA from a closed to an open complex

Question 7

describe the prokaryotic promotors

Answer 7

• Sigma factor recognises the -35 and -10 motifs common to prokaryotic promoters and enables RNA polymerase to make stable contacts with DNA.
• The region at -10bp called the pribnow box, there is also a region at -35 base pairs.
• The first nucleotide that is copied and transcribed into an RNA molecule will be +1. Nucleotides upstream of +1 will have a negative number.

– Binding of the sigma factor to the part of the promotor that will then recruit the RNA polymerase to the region of the promotor and start site of transcription.

Question 8

what is a consensus sequence

Answer 8

the sequence that you are most likely to find if you are studying a new promotor. It is the calculated order of most frequent residues found at each position in a sequence alignment.

Question 9

what is meant by the ability of the sigma factor and tfII to recruit RNA polymerase is generic

Answer 9

• it occurs at every promotor

- it does not account for the ability to vary the level of transcription

Question 10

what are regulatory transcription factors

Answer 10

• regulatory changes are mediated by regulatory TFs.
• •In both prokaryotes and eukaryotes they function to dramatically alter the level of recruitment of RNA polymerase and/or its ability to initiate transcription
  •Additionally, in eukaryotes they can influence local chromatin structure
• they recognise and binds to specific DNA target sequences, and do not need to unwind the DNA to see their target.

Question 11

what is a prokaryotic transcriptional switch

Answer 11

The lac operon

Question 12

give 3 examples of a eukaryotic transcriptional switch

Answer 12

–Oestrogen-responsive transcription
–Tissue-specific transcription (b-globin)
–A complex regulatory circuit (cell cycle)

Question 13

define an operon

Answer 13

clusters of genes that share the same promotor and are transcribed as a single large mRNA that contains multiple structural genes. a genetic regulatory system

Question 14

what is the on/off switch in a lac operon and what tf binds to it

Answer 14

operator - repressor.

Question 15

what is the lac operon in ecoli designed to do ?

Answer 15

breakdown lactose.

Question 16

what is the function of the lac operon

Answer 16

In bacteria, when there is a glucose and lactose, they always prefer to utilise glucose as a source of energy, as it has more energy. so therefore, if cells are in the presence of both glucose and lactose, the lac operon would be silenced and will not utilise lactose.

Question 17

what happens if there are low levels of glucose

Answer 17

if there are low levels of glucose, bacteria will try to utilise lactose if it is present in the media. In response to the absence of glucose/ low glucose levels, the CAP protein becomes active and binds to the promotor of the lac operon.

Question 18

what happens in the absence of lactose and low glucose levels

Answer 18

The lac repressor is another transcription factor that binds to this DNA sequence in the promotor. This gene is not expressed because it would be a waste of energy to express these enzymes if there is no lactose available. It is silenced as the cap protein which intends to recruit the sigma factor and RNA pol II but cannot transcribe the RNA molecule because of the lap repressor.

Question 19

what happens in the presence of lactose

Answer 19

In the presence of lactose, the lactose can bind to the protein and as a result the lac repressor is released from binding the DNA. This allows the cap to recruit the RNA polymerase and express the genes which will utilise the lactose as a source of energy.

Question 20

when is the cap protein active

Answer 20

low glucose concentrations

Question 21

+ Glucose, +lactose –>

Answer 21

OPERON OFF because CAP not bound

Question 22

+glucose, -lactose –>

Answer 22

OPERON OFF both because lac repressor bound and because CAP not bound.

Question 23

-Glucose, -lactose –>

Answer 23

OPERON OFF because lac repressor bound.

Question 24

-Glucose, +lactose –>

Answer 24

operon on – RNA is transcribed.

Question 25

what are the properties of transcriptional switches in eukaryotes ?

Answer 25

• Regulatory sequences recognise their target sequences by interacting with the DNA
• They do not have to unwind the DNA double helix to see their target
• Therefore, an intact DNA molecule can present information to the cell.

Question 26

what is the regulatory element

Answer 26

needed to regulate recruitment of RNA polymerase.

Question 27

how do regulatory transcription factors activate transcription ?

Answer 27

Eukaryotic regulatory transcription factors activate transcription by stimulating recruitment of general transcription factors and RNA polymerase.

Question 28

explain how steroid hormone signalling can regulate transcription

Answer 28

• Steroid hormones are lipophilic which means they can cross membranes and do not need a receptor
• Inside cells, the receptor for the steroid hormone is located in the cytoplasm,
• As a result of the steroid hormone binding to the receptor proteins, it changes the confirmation of the proteins and are competent to be translocated into the cell nucleus.
• In the cell nucleus, the hormone receptor complexes will now bind to specific regions in the promoters of the genes that will be transcribed in response to their binding.
• This results in the transcription into an Mrna molecule which is transported to the cytoplasm where the hormone receptor responsive genes are translated.

Question 29

what are the steps in oestrogen controlled portein synthesis

Answer 29

oestrogen crosses cell membranes and it will bind to the free oestrogen receptor complex in the cytoplasm, because of this binding the complex will translocate into the nucleus. The complex will start scanning all the genomic DNA for oestrogen responsive elements that are upstream of the oestrogen responsive genes.
As a result of this binding, it will recruit TFIID, TFIIB and this will recruit very efficiently RNA pol II.
All the genes that contain the sequences in their promoters will be transcribed.

Question 30

what is tamoxifen

Answer 30

drugs used in the treatment of breast cancer. It competes with oestrogen for binding to the oestrogen receptor. When it binds, it is unable to recruit RNA pol II to the oestrogen responsive genes and the expression of the gene will be silenced.

• Breast cancer dependent on the presence of oestrogen for growth. This is because some of the oestrogen responsive genes code for proteins involved cell proliferation. Inhibiting this process in breast cancer, inhibits cell proliferation

Question 31

describe the tissue specific transcription of the beta globin gene.

Answer 31

Beta globin gene, in the promotor has binding sites for transcript factors GATA-1, (3x). In the enhancer sequence, located distally to the transcriptional unit, it also has a high concentration of sequences which can be recognised by GATA-1. (4x)
Gata-1 is only expressed in cells that will be differentiated into rbcs, therefore we have a high expression of Beta globin gene only in RBCs because they are expressing the GATA-1 transcription factor. It is not expressed in other tissues because they don’t express the GATA-1 transcription factor.

The promotor also binds to ubiquitous factors (CP1, NF1, SP1) which induce the expression of the beta globin gene when the blood cells start to differentiate to RBC, leading to a very high expression in rbc due to GATA-1

Question 32

how are the phases in the cell cycle regulated

Answer 32

• G1 phase is regulated by the mitogenic signal- signal that tells the cell to start cell division.
• In the cycle, s, g2, and m phase are insensitive to regulation by external signals/ Stimuli

Question 33

How is the G1 phase regulated at the level of transcription?

Answer 33

Transitions through the cell cycle are regulated by CDKs which target transcription factors by phosphorylation. Key event in G1/S transition is the transcriptional activation of genes that encode proteins involved in DNA replication.

Question 34

what are the genes required for entry to the s phase and how are they regulated.

Answer 34

• The promotors for G1/S transition genes are activated by a factor called E2F.
• Genes code for proteins that are required for the synthesis of DNA eg DNA polymerase, DHFR, ribonucleotide reductase, thymine kinase – enzymes that are responsible for producing enough nucleotides for dan synthesis. Only expressed in the s phase.

-tatabox and e2f sites

Question 35

how are the genes that are required for entry to the s phase repressed.

Answer 35

• E2F activity is repressed in G0 and early G1 by the product of the retinoblastoma gene (pRB)
• Retinoblastoma gene is a tumour suppressor gene. When mutated, it causes tumours.
• Retinoblastoma binds to E2F, as a result it forms a complex at the promotor of the genes. This complex is not competent for binding and recruiting rna pol II, therefore the gens are not expressed.
• If the cells receive mitogenic stimulus, it will activate the cdks. The kinases are enzymes that phosphorylate the substrate proteins, and one of the substrates of the ckds that’s stimulated by the mitogenic stimuli is retinoblastoma protein.
• The cdks phosphorylate the retinoblastoma protein, which can now no longer bind to E2F and is released from the E2F.
• E2F WILL BE ABLE TO RECRUIT RNA PL II TO ACTIVATE THE EXPRESSION OF THE GENES.

Question 36

what is retinoblastoma and why is it a common target in cancer.

Answer 36

• Retinoblastoma inhibits E2F expression of genes that will allow the cells to proceed from the G1 to S phase. Therefore cells not dividing
• If received mitogens, the retinoblastoma is phosphorylated and it will detach from EF2 allowing the cells to progress through the cell cycle.
• Retinoblastoma protein is either not expressed or is actually mutated  it cannot bind to E2F  e2F is constantly active so the cells are always undergoing G2 specialisation and dividing  tumours
• Viral infection can transform the cells – the cells become cancerous. Viruses express viral proteins that compete for binding to the retinoblastoma protein. – rb unable to bind to E2F because it cannot be supressed.

Question 37

The recognition of promoters is mediated by initiation factors. What are these factors in prokaryotes?

Answer 37

→It is the sigma factor, which recognises the -35 and -10 motifs common to prokaryotic promoters.

Question 38

The recognition of promoters is mediated by initiation factors.

What are these factors in eukaryotes?

Answer 38

It is the TF2 basal transcriptional machinery (TF2A, TF2B, etc.).