Transcriptional circuits in prokaryotes

Question 1

What is a transcriptome?

Answer 1

→It is the segment of a genome that is transcribed.

Question 2

What are the 4 levels of gene transcription?

Answer 2

→Transcribed abundantly (if on in every cell, called a housekeeping gene)

→Transcribed rarely - Transcribed only in certain tissue cells (tissue-specific)

→Transcription is induced via a stimulus (turning it from a no/rare transcript to an abundant transcript)

Question 3

What are enhancers?

Answer 3

→They are sequences of DNA (not immediately adjacent to where transcription starts)

→act to enhance the recruitment of RNA polymerase to a promoter.

→They can reside in the 5’, 3’ or even the introns. They are very strong binding sites for specificity factors.

Question 4

what happens when RNA is recruited?

Answer 4

→In both eukaryotes and prokaryotes, RNA polymerases can’t make stable contacts with DNA, they slide along.

→Once stably recruited, the RNA polymerases convert from a closed complex to an open complex.

Question 5

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

Answer 5

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

Question 6

The recognition of promoters is mediated by initiation factors.

What are these factors in eukaryotes?

Answer 6

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

Question 7

what are regulatory transcription factors?

Answer 7

→ sequences that bind promoters and help recruit general transcription factors.

Question 8

List the known transcriptional switch in prokaryotes (1) and eukaryotes (3).

Answer 8

→PROKARYOTES: - the lac operon

→EUKARYOTES: - oestrogen-responsive transcription - tissue-specific translation (beta-globin) - a complex regulatory circuit (cell cycle)

Question 9

how does the lac operon work?

Answer 9

→E.coli prefer using glucose as an energy source, but when glucose is absent they can use lactose instead.

→The Lac Z gene codes for an enzyme that cleaves the lactose into simpler sugars.

→Lac Y codes an enzyme that is used for the absorption of lactose.

→When there is no lactose available then the cell will not transcribe the Lac genes because it is energetically expensive.

The lac repressor protein binds to the promoter region before the start site, the RNA polymerase will not be able to transcribe the genes as the repressor is blocking the RNA from moving forward.
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Question 10

what are inducible genes?

Answer 10

genes which are transcribed in response to stimuli.

Question 11

what do position and orientation independent mean?

Answer 11

→Position independent means that the enhancer can be cut out and be put in another place and it will still work.

→orientation independent so the enhancer can be reinserted backwards and it will still function properly.

Question 12

what do steroid hormones do and how do they do this?

Answer 12

→ steroid hormones control protein synthesis and they do this with the use of transcription factors

Question 13

what are the steps in estrogen controlled protein synthesis?

Answer 13

→Estrogen like all steroid hormones is lipid soluble so it crosses the cell membrane via diffusion and enters the cytoplasm.

→Estrogen binds to a receptor called ERα to form a hormone-receptor complex. This complex is now a transcription factor.

→The active transcription factor diffuses into the nucleus via the nuclear pore.

→ In the nucleus the transcription factor scans the DNA until it finds an estrogen responsive DNA sequence and binds the DNA promoter, upstream of RNA polymerase.

→This binding stimulates RNA polymerase to transcribe genes and so stimulates protein synthesis.

Question 14

what is tamoxifen and how does it work?

Answer 14

→ it competes with estrogen to bind to the ERα receptor.

→tamoxifen-receptor complex does not become a transcription factor and cannot enter the nucleus so cell proliferation is stopped.

Question 15

what are ubiquitous factors?

Answer 15

transcription factors that are expressed in all cell types

Question 16

give an example of tissue specific transcription

Answer 16

→Beta globin is only expressed in red blood cells, the promoter region contains different sequences that binds to many different transcription factors to activate the RNA pol II.

→In the beta globin gene there is an enhancer which is at the 3’ end of the gene (downstream).

→There are ubiquitous factors (TFs that are expressed in all cell types)

→The binding of these are not sufficient to determine tissue specific expression.

→GATA 1 is a TF expressed in cells that will be erythrocytes.

→it binds to the promoter regions and enhancers to activate the transcription of the tissue specific genes.

Question 17

how are genes regulated during the cell cycle? specifically G1

Answer 17

→Genes are very tightly regulated during the cell cycle.
→If cells get a mitogenic signal they enter the G1 phase of the cell cycle.
→ Before the G1 phase there is a restriction point which ensures all the enzymes for synthesis are there.
→Once the cells have passed G1-S phase the cells are insensitive to their external environment and cannot be modulated again.
→The transitions to the different phases are regulated by cyclins and CDKs.

Question 18

what are genes that are needed during DNA synthesis and how are they transcribed?

Answer 18

→DNA polymerase, and thymidine kinase which increases the levels of thymidine in the cells

→transcribed due to the transcription factor E2F

→it recognizes specific DNA sequences called E2F sites.

Question 19

what does retinoblastoma do?

Answer 19

→G1 retinoblastoma prevents E2F from binding and activating transcription to produce proteins for the S phase.

→If a mitogenic signal occurs, it activates the CDKs which phosphorylates the retinoblastoma which changes shape and releases the E2F.

→it is then free to bind to the E2F sites on the DNA.