Topic 2 Flashcards
Gene expression control
Temporally and spatially
- developmental stages
- specialized cell types
- regulation is in all tissues
4 mechanisms of euk. gene expression
Transcriptional control
Processing control
Translation control
Post-translational
Inducible response in yeast
Gal 4 transcription factor activates the expression of the Gal1 gene in the presence of galactose
Inducible response in Drosophilia
Heat shock transcription factor (HSF) activates heat shock protein gene (HSP) in response to heat
Environmental regulation of gene expression
i.e. heat shock genes
DNA binding protein - transcription factors
recognizes specific DNA elements (sequences)
Active: Free transcription factor
Inactive: Bound transcription factor to DNA
Heat shock transcription factor
- Constitutively expressed
- Monomer to trimer transition (3 domains touch)
- Trimerization mediated by interactions between hydrophobic heptad repeats (7a.a. repeat)
Signal transduction mediated by cell membrane receptors (provide an example and explain)
- Peptide hormones (extracellular signal)
- water soluble
- Receptor protein
- Intracellular signalling proteins
External stimuli, convey information to intracellular targets to effect response
Hormone/receptor complex on membrane = active when bound
Signal is sent to the nucleus - therefore change in gene expression
TF binds gene
Steroid Hormones
-lipid soluble
-released in blood stream
-receptor protein reside in cytoplasm
-hormone/receptor complex move into nucleus to activate gene expression (this is the active transcription factor)
-TF (complex) binds HRE hormone respone elements in the promoter og the gene
= activation
Transcriptional Activator
-bind to target promoter DNA element
-interact with basal transcriptional machinery
interact with chromatin
-nucleosome free DNA
-histone modification complex = transcriptional activator + enhancer: move away nucleosome to allow chromatin to bind
-insulators do the opposite: prevent spreading of euchromatin - stay as heterochromatin
Basal promoter elements
- DNA seq elements
- bind basal and general TF
- where RNA pol II is recruited and aligned on template DNA
- i.e. proximal promoter region & TATA box core promoter region
Basal TF, general TF
- protein transcription factor
- Basal TF: control constitutive gene expression in proximal promoter region
- General TF: TATA binding protein (TBP) & TFIIs – recruit, align, bind RNA pol II
Enhancer
- DNA sequence elements
- bind activator (transcription factor)
- TF binding provides gene specific control of expression
Activator
- transcription factor
- bind to enhancer sequence
- regulate transcription
- inducible expression: HSF1, steroid hormone receptors
Pre-initiation complex formed by
- general transcription factors = pre-initiation complex
- basal TF help assemble
- RNA Pol II cannot directly bind to basal promoter region
Basal promoter region
- proximal promoter region - CAAT, GC (-50 - -200) binds basal TF
- core promoter region - TATA (-30) binds general TF
General TF
TFIID: TATA binding protein (TBP) and TAFs
TFIIA/B
TFIIF: recruit RNA polII
Enhancer/repressor
- DNA bindingsite
- orientation independent
- position independent (i.e. upstream, downstream, within introns)
Enhancer & promoter relationship
- bend DNA to communicate
- done so by the mediator
Deletion analysis: deletion of the enhancer/promoter
- you can identify enhancers by deleting portions of an enhancer
- the deleted portion is the enhancer for the specific tissue
Properties of Promoters
- DNA sequence
- function within short distance
- immediately upstream from initiation site (RNA Pol II)
- position dependent (nonfunctional if moved)
- orientation dependent: drives one direction
i. e. CAAT -80, TATA - 30
Properties of Enhancers
- DNA sequence
- function long distance
- upstream, downstream, within introns
- position independent (functional if moved)
- orientation independent: function normal or inverted
DNA binding domain
-functional domain of transcription factors
-i.e.
Helix - turn - helix
Zinc Finger
Transcriptional activation domain
binding other proteins required for transcriptional activation
Dimerization domain
-facilitates protein protein interactions
-i.e.
Leucine Zipper
Helix-loop-Helix
Ligand binding domain
sensing external signals i.e. hormone
Domain
folding functional module of protein with several motifs
Motif
secondary structure of protein; a pattern
Helix-turn-helix
- DNA binding domain
- C term binds DNA
- 3 a helices, separated by turn
- others - dimerization
Zinc finger
- DNA binding domain
- knuckles bind DNA
- 2 cysteine
- 2 histidine
- zinc ion joins Cys + His
- dimers also knuckle
Leucine Zipper
- protein dimerization
- dimerization between leucines
- leucine every 7a.a.
- dimerization orients nearby DNA binding domains
Helix - Loop -Helix
- protein dimerization
- 2 a helices
- dimerization interaction of helical regions
- DNA binding domain adjacent to the helix loop helix
Protein dimerization increases…
- complexity of DNA binding specificity
- two transcription factor monomers (a, b, ab)
- can bind a combination of DNA elements, recognizing
Cooperative binding
- different transcription factors together provide complex means of regulation of gene
- TF bind to
- DNA sequence within
- Promoter/Enhancer which regulate gene expression
Mediator complex
links activators with RNA Pol II
- bridge between transcriptional activators (what’s bound to the enhancer) and RNA Pol II
- stimulate RNA synthesis
- brings distal promoter/enhancer and bound factors close to basal promoter region by looping DNA assisting in forming pre-initiation complex
- multisubunit complex
- histone acetylase activity: maintain nucleosome free DNA
What method identifies which transcription factor responsible for regulating which gene via? (3 methods)
- Identify promoter sequence required for transcription. Deletion mapping with reporter plasmids
- Measuring DNA-binding activity by gel-mobility shift assay
- Demonstrate DNA binding activity invivo by chromatin immunoprecipitation ChIP
Reporter plasmids to determine transcription factor regulation of gene
-identify regions within promoter that are essential for transcriptional regulation
Reporter plasmids contain:
- ORF for traceable protein i.e. luc
- MCS where you will insert the promoter of interest; also where you will delete portions of the promoter
- translation initiation elements upstream of ORF
- poly-A signal
Deletion mapping
- reporter plasmid to determine which transcription factor responsible for REGULATION of gene
1. clone DNA of interest (promoter seq) in MCS
2. Delete portions of region
3. Transfect plasmids individually into cells (+/- treatment)
4. measure reporter activity
5. Identify elements important to location
i. e. CAAT/GC box promoter (where basal TF bind) and TATA box (generalTF) if deleted will decrease activity significantly - if activity increases, then you have deleted a negative regulatory element
Gel-mobility shift assay
- tells whether or not cells contain transcription factor CAPABLE of binding to the specific DNA sequence
1. radioactive DNA probe containing promoter sequence
2. mix probe with cell extract of treated (+/-)
3. separate unbound probe DNA from bound probe/protein complexes on nondenaturing polyacrylamide gel
4. Expose gel to X-ray to detect radioactivity
Gel-mobility shift assay: results of gel
Supershift: Probe (promoter) + Protein (TF) + antibody
(antibody confirms it is the probe and not a competitor)
Shift: Probe OR specific competitor + protein
Free probes: unbound probe, mutant/non-competitor DNA sequences
Chromatin immuno-precipitation ChIP
- tells if cells contain transcription factor BOUND to chromatin
- in vivo - on a dish
1. treat formaldehyde kill cells and cross link TF to DNA. Isolate chromatin and shear fragments
2. Incubate with antibody that binds to TF of interest
3. Result: immunopreciptitated (bound to immune) chromatin fragments. Chromatin fragments (unbound)
4. Reverse cross-links, purify DNA
5. AmplifyDNA fragments with fluorescent label (ChIPSequence)
6. Hybridize DNA to microarray with a known intergenic DNA (ChIP-chip)
Eukaryotic mRNA processing
- post-transcriptional control of gene expression
- primary to mature mRNA
- 5’ 7MG cap and 3’ polyA tail added (regulating stability and translational efficiency)
- introns spliced out
- UTR untranslated regions - regulate mRNA stability and translational efficiency
- exons spliced together (EXpressed)
Alternate splicing of mRNA
- post-transcriptional control of gene expression
- more than one polypeptide per single gene
- alternate splicing of exons (which exons are spliced out - various combinations)
Control of mRNA localization
-post-transcriptional control of gene expression
-localization of mRNA encoding proteins in a certain area (clustered) determines the development - differentiation
i.e. flies
posterior: oskar mRNA - development of germ cells
anterior: bicoid mRNA - development of head and thorax
i.e. b-actin
actin protein accumulates at edge of cell to move the cell forward
Cytoplasmic control of mRNA stability: circular
- post-transcriptional control of gene expression
- increases efficiency
- polyA tail bind to polyA binding protein I
- interacts with initiation factors that is bound to 7MG cap
- forms circular mRNA
- increases translation efficiency by aiding ribosome subunit recycling (binding to start site)
Cytoplasmic control of mRNA stability: controlled by
- polyA tail: the length controls the mRNA stability
- short-lived mRNA: 3’UTR AUUUA = deadenylating unstable
- long-lived mRNA: 3’UTR CCUCC = stabilizing
mRNA degredation: polyA tail
-deadenylation dependent mRNA decay: nuclease (deadenylase) pregressively shortens polyA tail
-polyA tail <30 = degraded
via P-bodies
Degradation
1. decapping + 5’ - 3’ degradation
2. exosome mediated 3’ - 5’
RNA interference
- post-transcriptional expression regulation
- microRNA (miRNA) produced from double stranded RNA
- each mRNA can bind many miRNA + protein
miRNA and siRNA biogenesis
- produce miRNA and siRNA from large pri-miRNA
- double stranded hairpin structure
- Drosha crops hairpin to produce pre-miRNA
- Dicer cuts dsRNA to fragments
- siRNA in protein unwound to produce RISC
- Argonaute protein unwinds one strand of RNA and degrades (now ss)
- fragmented small interfering RNA assemble with ribonucleotide particle
- RNA induced silencing complex (RISC) = single strand interfering RNA + ribonucleotide
- RISC targets mRNA complementary to interfering RNA
-RISC base pairs target mRNA
- Perfect base pair: mRNA cleaved, degraded
- Imperfect base pair: translation mRNA arrested, polypeptide synthesis repressed
The name of the two genes associated with processing miRNA
lin-14 and lin 4
- lin 14 encodes TF that controls development
- lin 4 controlls lin 14 expression
- lin 4 precurser trimmed, complimentary to lin 14 UTR
- lin 4 DOWN regulates lin 14 (suppresses translation)
