chapter 13 Flashcards
(22 cards)
Prokaryotes vs Eukaryotes
- Eukaryotic cells are compartmentalized (organelles), prokaryotic cells generally are not
- Transcription and translation are separate in eukaryotes
Many eukaryotes are multicellular, prokaryotes are unicellular
* Different tissues have different patterns of gene expression
* Different developmental stages require tightly regulated gene expression (different factors, such as environment, affect gene expression)
- Eukaryotic genomes are packaged in a way that influences gene regulation (chromatin & epigenetics)
Mechanisms of Gene Regulation in Eukaryotes
In the nucleus, before transcription
-Regulatory proteins (bind to DNA)
-Regulatory sequences (enhancers, silencers)
-Chromatic structure
-Alternative promoters
-Methylation (histone modification can silence/activate)
DNA in nucleus - RNA in nucleus - RNA out of nucleus - polypeptide - protein
Mechanisms of Gene Regulation in Eukaryotes
In the nucleus, after transcription:
Alternative 5’ capping (protects from nucleases - present everywhere in sweat… and other molecules), 3’ polyadenylation, and splicing (allows stability)
AAAAA-3’
Mechanisms of Gene Regulation in Eukaryotes
After Splicing
Small RNAs that influence mRNA stability
Other factors that influence mRNA transport and stability and the initiation of translation
Mechanisms of Gene Regulation in Eukaryotes
In the cytoplasm, after export of mature mRNA
Factors that influence the initiation of translation
Mechanisms of Gene Regulation in Eukaryotes
after translation
Posttranslational modifications (downstream processes may further fold protein, add regulatory tags)
Binding of regulatory molecules
Regulation of protein stability
Enhancers and Silencers
Enhancers and silencers are distal regulatory sequences
- They bind regulatory proteins and interact with proteins bound to promoter
- Enhancers/silencers can be upstream or downstream of the genes they regulate
Sonic hedgehog (SHH) Gene
discovered in Drosophila
Mammals have three hedgehog homologues, one is called sonic hedgehog (SHH) - this one is smaller/condensed
The SHH gene offers a good example of how enhancers regulate tissue specific gene expression hedgehog mutant normal fly larvae
Sonic hedgehog and Tissue-Specific Gene Regulation
How can one gene have such a variety of functions?
SHH gene in humans and mammals directs development of limbs, including fingers/toes, and brain organization
Two enhancers: one for limb cells, one for brain cells
The one for limb cells is 1 million bp upstream in the intron of another gene (further from sonic hedgehog gene)
The one for brain cells is near the transcription start site in SHH gene
Regulation of SHH depends on what transcription factors are expressed in each cell
Conservation of Enhancer Sequences
Enhancer sequences can be highly conserved among diverse organisms which suggests natural selection
Very aligned with different mammals (all share enhancer sequences), change could be drastic and lead to speciation events
Conservation of Enhancer Sequences
GAL4-UAS System
In yeast, transcription of genes in galactose metabolism is regulated by enhancer sequences (regulates galactose digestion, off if galactose is absent) - This system parallels lac operon
When galactose is the only sugar available, yeast induce transcription of four genes that import and break down galactose (modify galactose into some derivative of glucose)
-Each GAL gene has its own promoter
-Each GAL gene also has its own enhancer called upstream activator sequence (UAS or UASG)
-The GAL4 protein is continuously present in cells and activates transcription by binding to the UAS elements
-GAL4 is inactivated when bound to GAL80 - not repressor, directly binds to GAL4
GAL4-UAS System
absence of glucose and galactose
Absence of Galactose: Gal4 is bound by Gal80 and is unable to activate transcription
Galactose Present: galactose and GAL3 protein bind to GAL80, freeing GAL4
GAL4-UAS System is: Widely used in Drosophila transgenic lines as an “ON/OFF” switch for genes (trans gene expression)
* GAL4 gene inserted in fly genome
* Another gene downstream of UAS sequence inserted in fly genome
* GAL4 will express whatever gene is inserted downstream of UAS
Insulator sequences
located between enhancers and promoters of genes
- They can block enhancer activity towards a specific promoter and redirect it to another gene
(regulatory proteins bind to and prevent enhancer regulation/function)
Insulator and Enhancer Interactions
Insulators are thought to block expression of
promoters by directing the formation of loops in DNA through regulatory proteins
a) Enhancer activity helps initiate transcription
b) Insulator sequence blocks enhancer action and can
c) Redirect enhancer activity to another gene
d) A particular enhancer activates a gene in preference over a nearby enhancer whose action is blocked
e) Insulators may direct the formation of DNA loops that contain enhancers and the genes they activate
Differential Genomic Imprinting of Chromosome 11 in Humans
Maternal Chromosome: enhancer drives expression of H19 and insulator protein blocks IGF2 expression
Paternal Chromosome: methylation inactivates the ICR and blocks H19 expression; enhancer drives IGF2 expression
Chromatin Structure Regulates Gene Expression more examples
- Heterochromatin can silence gene expression
- Euchromatin can maintain activated gene expression
- Chromatin can be remodeled by enzymes that alter associations with DNA and nucleosomes: Methylation, demethylation, acetylation, deacetylation
RNA Interference (RNAi)
Post-transcriptional regulation of gene expression
Double stranded RNA gets processed by enzyme Dicer into 21-24 bp fragments
* microRNA (miRNA): dsRNA expressed within a cell
* Short interfering RNA (siRNA): dsRNA from external sources (e.g. virus) - foreign material
Fragments bind to a protein complex called the RNA-induced silencing complex (RISC)
One RNA strand gets discarded in RISC
The strand remaining in RISC is called guide strand (find specific mRNA sequence)
The guide strand complementary base pairs with mRNA, destroying it before translation, silencing gene expression
The guide RNA bound to RISC directs one of three gene- silencing processes (knock-down gene: organism cant transcribe gene - not mutating/cutting, instead you target and inhibit it):
- RISC guide RNA binds to mRNA through complementary base pairing and destroys it
- RISC guide RNA binds mRNA and prevents translation
- RISC complex directs chromatin modifying enzymes to the nucleus where transcription of selected genes are silenced
Evolution and Applications of RNAi
- RNAi is widespread in eukaryotes
- Thought to help organisms protect their genomes against transposable elements and viruses
- RNAi applied widely to silence expression of genes to determine phenotypic effects
Dnase I Hypersensitivity
- Regions that are sensitive to Dnase I digestion are called Dnase I hypersensitive sites
- These regions contain euchromatin (more active/open) and are transcriptionally active
- A method of detecting transcriptionally active areas in a genome
Chromatin Immunoprecipitation (ChIP)
- A method to isolate DNA bound by regulatory proteins of interest
- Can identify where certain transcription factors bind and regulate gene expression
- Formaldehyde cross-links regulatory proteins to DNA where they bind
- Antibodies specific to protein of interest precipitates chromatin
- DNA can then be released from protein and purified
Interesting Examples of Regulated Gene Expression
Embryonic development (Various Genes and Enhancers Coordinate the Body Plan
of Embryos, diff banding patterns due to expression of genes)
Caste changes in eusocial insects (In honeybees royal jelly is fed to all larval castes by ‘nurse bees’
* Drones (male bees)
* Sterile female workers (i.e nurses, foragers)
* Queens
Larvae that become queens are isolated in a special cell in the hive and fed royal jelly for longer periods of time. Consuming higher amounts of royal jelly leads to epigenetic changes since queens and workers can be genotypically identical. It is thought royal jelly, in large quantities reduces DNA methylation levels, activating expression of “queen genes”
Stress response (Heat shock proteins (Hsp’s) are a family of stress response proteins
Hsp’s involved in re-folding or degrading damaged protein. Respond to heat stress, cold stress, infection, radiation, toxins, etc. Regulated by transcription factor heat shock factor 1 (hsf1). Malfunction of Hsp’s associated with many diseases including cancer)
Circadian clock (Circadian clock worked out in Drosophila; similar in other animals and humans. Genes period (per) and timeless (tim) get transcriptionally activated by genes clock (clk) and cycle (cyk). Per and Tim, at highest concentration at night, inhibit Clk and Cyc/ Cryptochrome (Cry) a blue-light receptor inhibits Tim in the presence of light, allowing Clk and Cyc to upregulate per and tim genes. This produces a rhythmic cycle of gene expression where light synchronizes the cycle
