genetics 8 Flashcards
what are eukaryotic genes regulated by
- promotors - core and proximal
- up/down stream - silencers and enhancers
- introns and exons - splicing of introns
what do distal/proximal enhancers do
- bind to transcription factors
what do core promotors do
- general transcription factors - TATA box - RNA polymerase
what does the promotor compartmentalisation do
- CCCTC binding factor
- zinc-finger DNA binding protein
- form DNA loops with cohesion
what is the function of promotor compartmentalisation
- Enhancer-promoter interactions
- Insulation against non-specific regulatory signals
- Transcriptional activator
- Stable chromatin loops - differentiation
- CTCF loop formation/loss
what are the functions of transcriptional activators
- promotes binding of other regulators
- release DNA polymerase to start
- recruits DNA polymerase
- release DNA polymerase from pause
what are the functions of transcriptional repressors
- Competitive DNA binding
- Masking activation
- Interact with Transcription actors
- Recruit chromatin remodelling complexes
- Recruit histone deacetylases
- Recruit histone methyl transferase
what happens with positive effector mediated regulation
- transcription factor binds
- RNA polymerase recruitment
what happens with negative effector mediated regulation
- repressor protein binds
- prevents gene expression by blocking RNA pol binding or block RNA pol movement
what are operons
- group of related genes under the same promotor
- inducible or repressible
- many in bacteria
when is Iac operon in E.coli lactose metabolism turned off and on
off - presence of glucose
on - presence of galactose or inducer (PTG)
how is Iac operon turned off in the presence of glucose
- repressor (homo tetramer) binds to operator
- reduce RNA polymerase activity
how is Iac operon turned on
- lactose metabolite (allolactose) inhibits Iac I repressor
what happens to Iac enhancer when there is no/low glucose
- low ATP –> cAMP increases —-> metabolism of other sugars
- CAP-cAMP binds to promotor
- enhancer RNA polymerase binds
what does trp operon do
- five enzymes necessary for tryptophan biosynthesis
how is trp operon transcriptionally controlled
- repressor binds to operator
- loop termination of mRNA synthesis via leader gene
what happens when the histones are acetylated or ubiquitinated
- N-terminal (lys)
- reduces + ve charge
- disrupts DNA binding
when a chromatin is decondensed what happens
- actively transcribed
- accesible to transcription factors
what occurs in RNA polymerase II transcription
- Histones ahead ubiquitinated
- Removed by histone chaperones
- Inserted behind
what does the chromatin remodelling factor do
- alters nucleosome structure
what are high mobility group proteins (HMGP)
- associated with active chromatin
- modify DNA histone interaction
- recruit transcriptional activators
what does DNA methylation act on
- DNA methyl transferase
- cytosine
what can deletion mapping do
- delete genetic regions
- increase/decrease expression
- change phenotype
- identify function
how does DNase hypersensitivity assays work
- cut DNA
- fragments cloned/ sequenced
- identify areas of DNA loosely attached to histones
- active areas
what does DNase I do in hypersensitivity assay
- cuts ssDNA or dsDNA
- digests easy accessible regions
what does Mg2+ do in the hypersensitivity assays
- randomly cuts dsDNA strand independently
what does manganese (Mn2+) do in the hypersensitivity assays
- cleavers both DNA strands
- same site
- blunt ends or 1-2 nucleotide overhang
what does DNase hypersensitivity assays show us
- differences in active areas eg. cell type, tissues, conditions
what does recombinant receptor assays do
- fusion of promotor to reporter gene
what does recombinant reporter assays tell us
- expression levels
- time during cell cycle
- induction / repression
what are the common assays in recombinant reporter assays
Green fluorescent protein (GFP)
Luciferase assay - luciferase/luciferin
GUS assay - β-glucuronidase/X-Gluc, PNG or MUG
Blue-white screen - β-galactosidase/X-gal
what are the components of gene regulation
- transcription factors
- genera transcription factors
- RNA polymerase
- chromatin remodelling complexes
- histone modifying enzymes
what is regulatory proteins function
- activate or deactivate different genes so:
- respond to environment - food intake, temperature
- cell growth and division
- development - embryonic and muscle cells
- polarisation/ iocomotion
- cell-cell communication
what are the corticosteroids mode of action
- Bind glucocorticoid receptors (GR)
- Bind glucocorticoid response elements (GRE)
- Histone H4 acetylation
- Anti-inflammatory genes
what happens when there is a high does of corticosteroids
- anti-inflammatory gene expression
what does oestrogen receptor antagonists do
- prevent/delay reccurence of breast tissues
what does tamoxifen do
- competitively binds to oestrogen receptors
what is an example of a helix-turn-helix motif
- tryptophan repressor
- binds as dimer at palindromic site
- type 2 restriction enzymes cut sites are palindromic
what do cys-cys-his-his family do
- zinc finger motifs
- zinc holds alpha helix and beta pleated sheets together
- chains of zinc fingers
what do leucine zipper motifs do
- y shaped structure
- DNA binding and protein dimerisation
- leucines hold helices together
- DNA binding in major grove
what do DNA loop helix do
- two alpha helices connected by a loop
- short- dimerisation and long - interacts with DNA
- regulated by interacting partner
what is an example of a loop region
- p53
- tumour supressor protein
- major and minor groove
what do coactivators and corepressors do
- don’t bind directly to DNA
- bind to DNA binding proteins (premiscous)
- scaffolds for other proteins
what do intergenic regions do
- prevent harmful transcription
what happens when a cell is methylated
- high dividing cells —> higher expression
- low dividing cells –> lower expression
what occurs with CpG islands
-1000bp
- often not methylated
- fewer associated nucleosomes
- methylation —> gene silencing
what do iron response elements do
- binds to iron response element binding protein
- regulate iron metabolism
what does ferritin do
- helps to store iron
- IRE at 5’ of mRNA
- low iron –> IRP binds IRE –. prevents translation
- high iron –> IRP binds iron –> translation increases –> iron storage
what does transferrin- receptor do
- iron uptake
- IRE at 3’ on mRNA
- low iron —> IRP binds to IRE –> mRNA stable –> increase iron uptake
- high iron –> IRP binds iron –> mRNA degraded
