PM Eukaryotic Gene Regulation and Expression Flashcards
Why is regulated gene expression important?
- Genes in eukaryotic cells are differentially expressed during cell growth and in response to environmental cues
- Gene expression programmes in metazoan organisms also drive cell differentiation and organism development
- Dysregulated gene expression will disrupt these processes, causing uncontrolled cell growth
What is a TATA box and where are they found?
- TATA motif within a larger AT rich sequence
- Found in promoters of highly inducible genes, located 30 nt 5’ of the TSS
What are CpG islands and where are they found?
- Clusters of CpG dinucleotides
- C is typically methylated
- Non-highly inducible genes are transcribed at multiple sites from these islands
- Methylated sites cause repression, hypomethylated regions cause expression
How can the TSS of a gene be mapped?
- Viral reverse transcriptase can generate a ssDNA from an RNA template
- Short DNA primers are annealed to the ssRNA
- The DNA primer is extended 5’ to 3’ by RT until it reaches the 5’ end of the RNA
- Primer extension products are analysed by gel electrophoresis
Describe deletion analysis of promoter regions
- When you know the transcriptional start site of gene, regulatory regions within promoter regions can be defined by deletion analysis, using a reporter gene eg LacZ or luciferase (cell extracts from transformed cells are assayed for LacZ or luciferase activity)
- Define the ends of the regulatory regions; deletions from one end of the promoter or the other
- Making shorter fragments of promoter region
- Clone fragments into cell to see if it still works
- Deletions beyond a certain point reduce enzyme activity
Describe linker scanning mutagenesis of promoters
- Linker scanning mutagenesis of promoter regions can identify sequences that are important for gene expression
- Define a small region that is essential for gene expression
- Find the nucleotides within that sequence that are important
- Make a series of constructs with mutated DNA at different sites
- The mutants which have an impact on gene expression have mutations in the essential regions
What transcriptional regulatory elements are found in mammalian genes, and where are they in relation to the TSS?
- Enhancers: can be upstream of TSS, in introns or downstream of coding region
- Promoter-proximal element: ~200 nt. 5’ of TSS
- TATA box: ~30 nt. 5’ of TSS
- Gene (exons and introns)
What transcriptional regulatory elements are found in yeast genes, and where are they in relation to the TSS?
- UAS/URS: upstream of the TSS
- TATA box: ~90 nt. 5’ of TSS
- Gene
What is the TATA box binding protein?
A protein within TFIID which binds directly to the DNA, causing a bend and allowing other proteins to be recruited
What is the difference in transcriptional regulation between prokaryotes and eukaryotes?
- In prokaryotes, regulatory proteins directly target the polymerase
- In eukaryotes, regulatory proteins impact polymerase indirectly via the mediator complex and by altering chromatin structure
What is the difference between general and specific transcription factors?
Specific transcription factors interact with some promoters in specific sequences but not all, whereas general transcription factors interact with all promoters in all genes
What are regulons?
Groups of genes showing similar transcription profiles
How is coordinated regulation typically achieved?
Through the activity of sTFs that recognise promoter-proximal elements upstream of each gene of the regulon
Why are functionally linked genes regulated by regulons and not operons?
- In prokaryotes, genes are physically linked and can therefore be controlled by one TF
- However, genes in eukaryotes with similar functions are not linked physically and may be spread around in the genome
- These nevertheless require the same TFs
How can variation in transcriptional control be achieved?
Through combinatorial control: recruitment of different combinations of sTFs
What is GAL4 and where does it bind?
- GAL4 is required for the expression of genes that encode proteins that are necessary in order for the yeast to use lactose and binds to the UAS
- GAL4 has functionally separable DNA-binding & activation domains
Describe the yeast two-hybrid analysis of UAS and GAL4
- If UAS is recognised by GAL4, and this is placed upstream of a gene, this will recruit other proteins necessary for transcription and the reporter gene will be transcribed
- If GAL4 is removed, transcription will not occur
- However, if DNA-binding and activation domain are bound to proteins which can interact with each other, a chimeric reconstitutive GAL4 can be produced and the gene will be transcribed
What is the yeast two-hybrid used for?
To identify and analyse protein interactions
