Module 3--Regulation of gene expression Flashcards
What is promoter?
A length of DNA sequence “upstream” of the transcribed region
What is the function of promoter?
Regulates gene expression through interaction with proteins
Where is the RNA polymerase binding site in prokaryotes?
-35 & -10 regions of lac promoter (where sigma factor binds)
Where is the strand separation site?
-10 region (A=T rich sequence)
What is consensus sequence?
Ideal promoter sequence
What kind of promoter match the consensus sequence?
Strong promoter
Is lac promoter strong or weak under normal conditions?
Weak
What is the mechanism of the initiation of transcription?
- Different sigma factors compete to bind to core polymerase
- RNA polymerase slides along DNA
What conditions are required for lac operon promoter to be active?
- Cell needs energy
- Glucose not available
- Lactose is available
What is stringent response?
- Under good nutrition, sigma D binds to RNA polymerase (housekeeping genes–>growth)
- Under poor nutrition, sigma S (stationary phase/starvation) binds to RNA polymerase
(housekeeping genes suppressed–>maintenance)
What is alarmone (ppGpp)?
An intracellular signal molecule that is produced due to harsh environmental factors
What is the function of alarmone (ppGpp)?
It binds to RNA polymerase which reduces affinitiy of RNA polymerase for sigma D, so sigma S and other stress sigmas can compete for binding
-This reduces transcription of housekeeping genes and increases transcription of stress (other) response genes
Alarmore (ppGpp) is produced by:
RelA or SpoT
(RelA+uncharged tRNA when amino acid starvation;
SpoT-glucose when glucose starvation)
Alarmone (ppGpp) is degraded by:
SpoT (SpoT+glucose when glucose abundance)
What are the other regulators of sigma factors?
- Regulation of gene transcription
- Regulation of mRNA stability
- Inhibition of translation
- Inactivation by proteolysis
- Activation by removing inhibitory N-terminal amino acid extensions
- Inhibition by high affinity binding of anti sigma factors
What is cyclic AMP (cAMP)?
A specific glucose starvation signal
What is the function of cyclic AMP (cAMP)?
It binds to CRP (cyclic AMP receptor protein) when glucose is not available
What is allolactose?
A modified form of lactose
What is the function of allolactose?
It binds to lac operon repressor when lactose is available, which prevents inhibition of lac operon transcription
What is signal transduction?
Complicated protein modification networks & small molecule signals
What is the function of transcription factors (TFs)?
Interact with promoters to facilitate RNA polymerase binding
What is the cellular response to environmental factors?
1) Receptors
2) Signal transduction (communicating/integrating)
3) Transcription factors (TFs) (responding)
What is the mechanism of regulation by peptide hormones?
1) Hormone binds to a receptor protein in the membrane of its target cell
2) Hormone/receptor complex activates cytoplasmic protein
3) Activated cytoplasmic protein transduces a signal to the nucleus
4) The signal induces a transcription factor to bind to DNA
5) The bound transcription factor stimulates transcription
6) The transcript is processed and transported to cytoplasm
7) mRNA is translated into proteins
What is the mechanism of regulation by steroid hormones?
1) Steroid hormone enters its target cell and combines with a receptor protein
2) Hormone/receptor complex binds to a hormone response element in the DNA
3) Bound complex stimulates transcription
4) Transcript is processed and transported to cytoplasm
5) mRNA is translated into proteins
What is the induction of genes by heat shock?
Under heat stress, heat-shock transcription factor (HSTF) is phosphorylated and induces transcription
What are the pathways of gene regulation in longevity in eukaryotes?
- Good nutrition –> housekeeping genes –> growth/reproduction
- Poor nutrition –> starvation signalling –> stress genes –> longevity/suppressed reproduction
What are the longevity inducers?
- Dietary restriction
- Decrease in insulin signalling (cellular glucose deficit)
- AMP kinase (AMPK) signalling (energy depletion)
- Amino acid signalling (amino acid deficit)
- Decrease in mitochondrial function (energy depletion)
- Decrease in temperature (decrease in metabolic rate)
What and where is the core promoter in Eukaryotes?
It is assembly site of preinitiation complex containing basal transcription factors and RNA polymerase II, and is immediately upstream of transcription start
What does core promoter in Eukaryotes contain?
It contains a TATA box, AT rich sequence (site of strand separation)
What and where is proximal element in Eukaryotes?
It is site of binding for activator proteins and is immediately upstream of promoter
How can proximal elements lose their activity?
Moves further upstream by more than a few tens of nucleotides
How to conduct molecular genetic analysis of gene regulation?
Gene regulatory sequence is ligated to the protein coding sequence of GUS reporter gene
-If promoter is active, GUS is expressed and cell turns blue
How to define the boundary of the core promoter?
Progessive 5’ deletion until GUS reporter gene is not expressed
What and where is enhancer/distal element in Eukaryotes?
It is a site of binding for special transcription factors and is at upstream of promoter and proximal control elements
How to define the boundary of upstream regulatory elements?
By linking upstream elements to core promoter
How to enhance the activity of core promoter?
It is enhanced greatly by distal elements
Transcription begins when polymerase is:
Phosphorylated
What is the basal transcription factor assembly process?
- TFIID (TATA binding protein) binds to TATA box & bends DNA sharply
- Other basal factors assemble
- RNA polymerase II holoenzyme binds to TFIID
What is proximal element important for?
Important for constitutive expression of housekeeping genes
What does high level gene transcription need?
Enhancers/Distal elements
For non-housekeeping genes, why would transcription initiation be infrequent?
Gene has only promoter + proximal elements
What is the function of enhancers/distal elements/response elements?
Precise control of gene regulation
What is mediator complex and its function?
It bends DNA of regulatory region and brings distal elements to the promoter
What is special transcription factors?
Nearly always positive regulators
What is the structure of transcription factor?
- DNA binding domain: recognizes 4-8 bp sequences
- Dimerisation domain: most transcription factors are inactive as monomers
- Activation domain: interacts with subunits of mediator or RNA polymerase II
What happens if there are sequence changes in transcription factor genes?
Changes development and thus phenotype
Why changes in transcription factor genes do not lead to complete disorder?
Transcription factors coordinate the activity of many genes that function together
Why most natural gene variants do not change protein function, but only gene expression?
Changeing expression levels is easy, but changing protein function is difficult
Does the position of enhancer has effects on its activity?
No, effects of enhancers are independent of position; they may be upstream, downstream or in an intron
What is epigenetics?
Semi-heritable information encoded as molecular tags on the DNA and associated proteins (life experiences & developmental programs)
What is the structure of nucleosome?
It consists of 8 histone proteins + ~200 bp of DNA
What does access to DNA in vivo require?
Chromatin modification
How can nucleosomes be compacted tightly together?
H1 is required
What is the function of histone H3?
Regulates chromatin activity
How is histone H3 modified?
Acetylation and methylation
What is nucleosome in inactive state?
DNA condensed tightly and histones not easily be removed
What is nucleosome in active state?
Histones can be removed
How can histone be modified?
- Reversible acetylation
- Reversible methylation
- Epigenetic tags added to histone
What enzymes are used in reversible acetylation?
- Histone acetyl transferase (HAT): adds acetyl group
- Histone deacetylase (HDAC): removes acetyl group
What enzymes are used in reversible methylation?
- Histone methyl transferase (HMT): adds methyl group
- Histone demethylase (HDM): removes methyl group
Does H3K9 acetylation activate or inactivate gene?
Activate gene
What does H3K4 methylation do?
Enhances active site
Does H3K9 methylation activate or inactivate gene?
Inactivate gene
What are epigenetic tags and what do they do?
Records of the developmental/envrionmental experience of an organism:
- no change to nucleotide sequence
- semi-heritable/dynamic process
- can facilitate or inhibit normal transcriptional control
What leads to DNA methylation?
H3K9 methylation
What is DNA methylation?
DNA very tightly condensed = genes strongly off
What are the examples of methylation of repetitve DNA?
- Heterochromatin = centromeric and telomeric DNA (highly methylated and condensed)
- Dispersed repeats: viruses and transposons dispersed throughout the genome (inactivated by methylation, become reactivated when host under stress)
What is the effect of methylation in development?
- Methylation from previous generation erased prior to embryo implantation
- Remethylation coincides with developmental progression
How can diet cause methylation?
Mother’s diet during development can influence the epigenetics of child
What is DNA imprinting?
30-200 genes are differentially inactivated by methylation in gametes of males or females
What is penetrance?
Probability of a gene to be expressed
What affects penetrance?
- Methylation of genes
- Somatic methylation: environmental/developmental modification of penetrance
- Imprinting: sex-specific modification of penetrance
How is methylation of viruses & transposons?
- Methylation of transposons is maintained in somatic cells, gametes and across generations
- Demethylation: mobilization under severe stress
What are the types of methylation?
1) Dynamic gene regulation: erased in early embryogenesis, re-established during lifetime
2) Imprinting: erased and re-established early in gamete formation
3) Suppression of transposons (by piRNA signalling): maintained indefinitely, but erased under stress
4) X chromosome inactivation: cell-by-cell decision in early female embryo
What is X-chromosome compensation?
If males and females have different numbers of X chromosome:
-Gene expression must compensate for the difference in gene copy number
What is transgene?
Gene integrated into the genome in a genetic engineering experiment
What is co-suppression?
Normal gene and transgene are both suppressed, uses RNA interference as mediator of co-suppressioon
How does transgene suppression work?
- Suppressed transgene is methylated: transcription is inhibited
- Transcripts are degraded
What is RNA interference and how does it work in all Eukaryotes?
dsRNA processed into siRNA (=small interfering RNA), which directs proteins to degrade mRNA transcript and suppresses gene expression
What is micro RNA (miRNA) and its function?
It is single-stranded RNA (ssRNA) and binds to 3’ end of mRNA transcript which blocks translation
What is the mechanism similar to siRNA and miRNA?
- RNA cut into small pieces
- Combines with a RISC (RNA induced silencing complex)
- RNA directs RISC to target nucleic acid, in which target identified by complementary base-pairing
What is the pathway of RNA interference?
Dicer processes dsRNA into siRNA
1) Double stranded RNA cut into small fragments
2) Protein complex assembled with small RNA
3) RNA delivers RISC to complementary mRNA sequence
What are the examples of silencing mechanisms?
- mRNA degradation (siRNA)
- Inhibition of translation (miRNA)
Overview of small RNAs
- RNA processing
- Silencing complex assembly
- Degradation
- Translation inhibition
- Chromatin remodeling
What is parallel regulatory system?
- RNA fragments produced in parallel with protein synthesis, which are byproducts of mRNA processing
- Indicator of gene expression
Summary of RNA function:
- Defence against viruses
- Suppressor of transposons
- Regulator of development
- Coordinator of chromatin remodeling
- Systemic coordinator of gene expression
