Transcriptional circuits in prokaryotes and eukaryotes Flashcards
What is the transcriptome? (3)
- Most of the eukaryotic genome is never transcribed; only about 50% of the prokaryotic genome is transcribed
- The segment that is transcribed is referred to as the transcriptome
- Only a fraction of the transcriptome is transcribed at any one time
What 4 genes can be transcribed at different levels? (4)
- 3 types of genes.
- Gene 1 gives rises to many copies of RNA therefore called abundant transcript, if found in each cell type they are called housekeeping genes as they produce specific proteins in every cell type e.g. glycolytic enzymes uses glucose to generate energy. E.g RNA polymerase.
- Gene 2 for which not much protein is required, low levels are produced so called rare transcript.
- Gene 3 only found in some genes e.g. no transcript in cell one but in cell two it is highly abundant as they code for tissue specific genes e.g. globin in RBC but no other cell types.
- Genes can be ‘inducible’ genes. So, if a stimulus is induced this can lead to the transcription of more than one gene making an abundant transcript.
Transcription requires recognition sequences in DNA that lie outside of the transcribed region…
- The sequence immediately 5’ to the region to be transcribed is called a “promoter”.
- Promoters recruit RNA polymerase to a DNA template.
- RNA polymerase can only move one way.
What are enhancers? (4)
- 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”
- Enhancers are position independent manner, so it can be in any position of a gene and it will work. Can be found up/down stream of a gene. Also work in an orientation independent manner so if the enhancer was cut out and flipped around it can be put back and still function. This will not work with promoters as RNA polymerase will work in opposite direction.
Describe the recruitment of RNA polymerase to the promoter region (5)
- Neither prokaryotic nor eukaryotic RNA polymerases make stable contacts with DNA – they slide along the duplex without being able to efficiently recognise promoters
- Recognition of promoters is mediated by initiation factors.
- Predominantly “Sigma factor” for prokaryotes.
- The TFII basal transcriptional machinery for eukaryotes.
- Once stably recruited to DNA, RNA polymerase is able to convert from a closed to an open complex.
Describe prokaryotic promotors (4)
- The first nucleotide encoded into RNA is called +1 (initiation site) and then so on. Nucleotides before this are not copied into RNA and are numbered in negative numbers.
- In bacteria RNA polymerase does not bind to this sequence. This sigma factor binds and then RNA polymerase is recruited.
- Consensus sequence – a sequence of DNA having similar structure and function in different organisms. If you align all promoters there is a common sequence e.g. TATA (TATA box).
- The Pribnow box (also known as the Pribnow-Schaller box) is the sequence TATAAT of six nucleotides (thymine, adenine, thymine, etc.) that is an essential part of a promoter site on DNA for transcription to occur in bacteria.
Describe regulatory transcriptional factors
• The ability of sigma factor and TFII to recruit RNA polymerases to promoters are generic – i.e. they happen at every promoter. o This does not account for the ability to VARY the level of transcription from a promoter. • Regulatory changes are mediated by a different class of transcription factors = Regulatory Transcription Factors. • 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. • Regulatory factors recognise their target sequences by interacting with the DNA. o They do not have to unwind the DNA double helix to see their target. o Therefore, an intact DNA molecule can present information to the cell.
Give 4 examples of prokaryotic and eukaryotic transcriptional switches
Transcriptional switches
Prokaryotic:
• The lac operon
Eukaryotic:
• Oestrogen-responsive transcription
• Tissue-specific transcription (b-globin)
• A complex regulatory circuit (cell cycle)
Describe the Lac Operon
• Enzymes catalysing metabolic reactions are synthesised at constant rates, but enzymes that are only needed under specific conditions are synthesised at varying rates according to the cell’s needs.
• E. coli normally metabolises glucose, but if absent and lactose present, lactose is metabolised.
• The lac operon is a length of DNA containing:
o Control sites: the operator region, lacO, and the promoter region, P.
o Structural genes: lacZ (codes for lactose permease - an enzyme allowing lactose to enter the bacterial cell) and lacY (codes for Beta-galactosidase - hydrolyses lactose → galactose).
o ○ Regulatory gene, I, coding for LacI (a repressor protein).
• When the regulatory gene is expressed: LacI is produced → binds to the operator region → prevents RNA polymerase from binding to the promoter region → lacY and lacZ aren’t produced → lactose cannot enter the cell and cannot be hydrolysed (as neither enzyme is present).
• When lactose is present (and all glucose has been hydrolysed): lactose binds to the LacI repressor protein → conformational change of repressor protein → no longer binds to the operator → RNA polymerase can bind to the promoter region → lacY and lacZ can be expressed → enzymes synthesised → lactose can enter the bacterium and can be metabolised.
Describe steroid hormone signaling
- Are lipid soluble – lipophilic.
- They pass through the lipid component of the cell membrane and bind to the steroid hormone receptors to form a hormone-receptor complex. The receptors maybe present in the cytoplasm or the nucleus depending on the hormone.
- The hormone-receptor complex formed acts as a transcription factor which in turn facilitates or inhibits the transcription of a specific gene.
- e.g. oestrogen/testosterone.
- Eukaryotic regulatory transcription factors activate transcription by stimulating recruitment of general transcription factors and RNA polymerase.
- Oestrogen binds to oestrogen receptor complex which then binds to TATA box.
- Lead to recruitment of TFII and RNA polymerase II, leading to start of transcription.
- Studying oestrogen lead to the discovery of Tamoxifen for treatment of breast cancer. It acts as a competitive inhibitor. So, RNA polymerase II is not recruited, and tumour growth is inhibited.
Describe Tissue-specific transcription (β-globin)
- Ubiquitous factors - Transcription factors that are found in all cell types e.g. TFIIA, TFIIB/D/E/F/H.
- GATA-1 is a TF that only expressed in RBCs, the presence of this allows binding to promoter and enhancer regions.
Describe regulation of the cell cycle
Transitions through the cell cycle are regulated by cyclin-dependent kinases
• Key event in G1/S transition is the transcriptional activation of genes that encode proteins involved in DNA replication.
• CDKs target transcription factors.
- The promoters for G1/S transition genes are activated by a factor called E2F.
- E2F recognise a specific DNA site.
- E2F activity is repressed in G0 and early G1 by the product of the Retinoblastoma gene (pRB).
- The mitogenic stimulus will active cDk this kinase will phosphorylate the retinoblastoma gene, changing shape so it can no longer bind to E2F. Then E2F can now bind and recruit RNA polymerase II.
- Rb is a common target in cancer, eye cancer.
