Chapter 12-13 Flashcards
(84 cards)
1
Q
Biology central dogma
A
DNA -> transcription -> RNA -> translation -> protein
Gene expression = transcription + translation
2
Q
How does epigenetics regulate gene expression
A
histone modification up-regulates (activates) or down-regulates (silences) gene expression
3
Q
When is regulation done in pro vs. eu
A
Pro
- mostly during transcription
Eu
- post-translational modifications
4
Q
Constitutive transcription
A
constant expression of genes
- no regulatory control
5
Q
Regulated transcription
A
expression only occurs under certain conditions
(ex. food scarcity)
6
Q
Posttranscriptional regulation
General
A
After mRNA is synthesized, its abundance can be modified to influence the amount of protein translated
7
Q
Thermostat analogy (regulated expression)
A
- waste of energy for thermostat to always be ON
- so keep OFF by default unless too hot/cold = turn ON
this is how many genes are regulated
8
Q
Negative control of transcription
A
involves a repressor protein binding to a regulatory DNA sequence, preventing transcription of
a gene or genes
9
Q
Positive control of transcription
A
involves binding of an activator protein to a regulatory DNA sequence and initiating transcription of a gene or genes
10
Q
Negative control
A
Repressor = no trans
Corepressor causes repressor to bind = no trans
Inducer removes repressor = trans
11
Q
Positive control
PEI
A
Activator = trans
Effector causes activator to bind = trans
Inhibitor prevents activator from binding = no trans
12
Q
Allosteric change
hint: not between genes
A
when interactions between proteins change their conformation and function
13
Q
Lac operon
A
a commonly studied bacterial gene regulation system in E. coli
14
Q
Lac operon - negative control
think normal vs when lactose present
A
a repressor binds to operator sequence, preventing transcription
- Prevents synthesis of enzymes involved in lactose metabolism
- Only activates when lactose is present in environment
15
Q
Lac operon - positive control
A
transcriptional elements more active when glucose is absent
16
Q
What causes up-regulation of lactose metabolizing genes
presence/absence
A
Presence of lactose & absence of glucose
17
Q
Preferred energy for E coli
A
- prefer glucose
- lactose can be used if glucose absent from the environment
18
Q
Lactose breakdown for E coli (2 things)
A
- transport using permease transport protein
- lactose breakdown into glucose and galactose through beta-galactosidase
= glucose present for energy
19
Q
Role of beta-galactosidase
A
breaks lactose down into three things
1. glucose
2. galactose
3. allolactose
20
Q
Allolactose
A
from lactose
acts as an inducer compound that binds to the repressor protein, removing its inhibition from the lac operon
21
Q
What is an operon
A
a cluster of genes undergoing coordinated transcriptional regulation by a shared regulatory region
22
Q
Polycistronic mRNA
A
a single messenger RNA (mRNA) molecule that contains the genetic information for multiple proteins, often found in prokaryotes
ex. the lac operon, trp operon
23
Q
The lac operon - parts
A
lacl
lactose operon
= promoter region (CAP binding site, lacP, lacO) + structural gene region (lacZ, lacY, lacA)
24
Q
Lacl
where + binds to what
A
regulatory gene that is adjacent but not part of the lac operon
lacL repressor binds to the operator sequence (lacO) and blocks transcription of the lac operon
25
LacP
| ____ binds to this
promoter sequence
RNA polymerase binds to this
26
LacO
operator
27
LacZ
beta galactosidase gene
28
LacY
permease gene
29
LacA
transacetylase gene
Transfers acetyl groups. Thought to protect against harmful by-products of lactose metabolism
30
Positive Control: CAP-cAMP Complex
CAP-cAMP complex activates transcription of lac operon
Positive control occurs at the CAP-cAMP binding region of the lac promoter
* CAP = catabolite activator protein (CAP)
* CAP upregulates transcription by RNA polymerase
* cAMP = cyclic adenosine monophosphate
* cAMP is synthesized from ATP by adenylate cyclase
31
Glucose ________s adenylate cyclase activity
and effect
inhibits
Low [glucose] → high [cAMP] → CAP-cAMP complex forms
High [glucose] → low [cAMP] → CAP-cAMP complex does not form
32
Presence of Glucose; Absence of Lactose
what type of expression is LacL repressor gene under (constitutive vs regulated gene expression)
LacL makes repressor
* Repressor bound to LacO
* cAMP low
* No transcription
of lac operon (lacZ/Y/A)
LacL repressor is constitutively expressed and represses transcription of the lac operon
33
Absence of Glucose; Presence of Lactose
| repressor removed how?
* Repressor removed
* cAMP high
* Transcription of lac
operon
allolactose is produced from lactose = acts as an inducer to stop repression of the lac operon
RNA polymerase binds to the promoter and transcribes more than basal level
34
Presence of Glucose and Lactose
* Repressor removed
* cAMP low
* Low transcription of
lac operon
lac operon is transcribed at basal level = allolactose production from lactose (when present)
35
If allolactose is made from beta galactosidase, how does the cell ever have the allolactose that is needed to induce beta galactosidase?
There is always a small amount of beta-galactosidase expressed, even in the absence of allolactose (“basal” expression)
36
Loss of function mutation in lacI (repressor gene)
leads to constitutive expression of lac operon
37
Operator constitutive mutation
prevents wild-type repressor from
binding, leading to constitutive expression of lac operon
38
Super repressor mutation
| No inducer
prevents inducer from suppressing repressor, leading to no transcription of lac operon
39
trp operon
what's it made of
polycistronic
Another operon in E. coli that contains five genes involved in synthesis of amino acid tryptophan
* trpE, trpD, trpC, trpB, trpA
Regulatory sequences are also present upstream
* Promoter: trpP
* Operator: trpO
* Leader (attenuator): trpL
A repressor gene is expressed outside of the operon but the protein
regulates operon
* trpR
40
The order of trp operon genes ...
corresponds to sequential steps of tryptophan synthesis
P, O, L, E, D, C, B, A
41
trp operon regulated by tryptophan; tryptophan present
when tryptophan present, transcription levels are low
but not zero = basal transcription
repressor active
42
Tryptophan is a ________
corepressor
43
trp operon regulated by tryptophan; tryptophan absent
when tryptophan absent, high transcription
inactive repressor
44
Attenuation of trp operon
is a second mechanism that controls the expression of the operon
level of tryptophan influences level of operon expression
45
Bacterial transcription + hairpin
transcription and translation coupled in the cytosol
secondary RNA hairpin structure formed and terminates transcription in bacteria
hairpin weakens association with RNA polymerase and mRNA
46
Attenuation of trp operon
| Hairpin and trpL
Hairpin structures from trpL mRNA also regulate operon
trpL precedes the other trp genes in the operon
in bacteria as trp genes are transcribed, they can immediately begin translation
trpL has two tryptophan amino acids in a row
Early in transcription, the mRNA of the trp operon forms multiple hairpin structures that
determine whether transcription continues or not
47
Two types of hairpins
if hairpin with 3/4 together
- hairpin repeat between region 3 and 4
- transcription stops early and most of the trp operon is not transcribed
- rapid translation of trpL due to lots of tryptophan causes this hairpin = termination of transcription
if hairpin with 2/3 together;
- hairpin repeat between region 2 and 3
- whole operon transcribed
- slow translation of trpL due to lack of tryptophan causes this hairpin = continuation of transcription
48
Heat shock response
Transcriptional activation triggered by the presence of an environmental stress, such as heat
49
HSR in E Coli
E. coli display a heat shock response at temperatures higher than 37 deg
* Use alternative sigma factors to activate heat shock response genes
Sigma factor required for transcription initiation
* Bacteria have different sigma factors for transcribing different classes of genes
* Heat shock response genes include chaperone proteins, which help fold or
degrade proteins denatured by heat
At normal temperatures, a subunit of RNA polymerase called sigma 70 is used for normal transcriptional processes
At higher temperatures, a different sigma factor gets translated (sigma 32) and is incorporated into RNA polymerase and drives expression of heat shock genes
50
Prokaryotes vs. eukaryotes
pro
- cells not compartmentalized
- unicellular
eu
- cells compartmentalized
- separate transcription and translation
- multicellular
- genomes packaged in a way that influences gene regulation (chromatin and epigenetics)
51
Different tissues have _____________ patterns of gene expression
different
52
Different developmental stages require ...
| something about gene expression
tightly regulated gene expression
53
Gene Regulation in Eukaryotes - before transcription
in nucleus
* regulatory proteins
* regulatory sequences (enhancers, silencers)
* chromatin structure
* alternative promoters
* methylation
54
Gene Regulation in Eukaryotes - after transcription
in nucleus
* alternative 5' capping
* 3' polyadenylation
* splicing
55
Gene Regulation in Eukaryotes - after splicing
exiting the nucleus
* small RNAs that influence mRNA stability
* other factors that influence that influence mRNA transport and stability and the initiation of translation
56
Gene Regulation in Eukaryotes - after export of mature mRNA
in cytoplasm
* factors that influence the initiation of translation
57
Gene Regulation in Eukaryotes - after translation
in cytoplasm
* posttranslational modifications
* binding of regulatory molecules
* regulation of protein stability
58
Enhancers and silencers
| Bind to
Interact with
Located
distal regulatory sequences
bind regulatory proteins
interact with proteins bound to a promoter
can be upstream or downstream of the genes they regulate
59
Sonic hedgehog gene (SHH) - discovery
* A critical embryonic development gene called hedgehog was discovered in Drosophila in the
1970s
* Mammals have three hedgehog homologues, one is called sonic hedgehog (SHH)
* The SHH gene offers a good example of how
enhancers regulate tissue specific gene
expression
60
Sonic hedgehog gene (SHH) - tissue specific regulation
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
* 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
61
Conservation of enhancer sequences across diverse organisms
sequences highly conserved
suggests action of natural selection
ex. enhancer of the interferon gene, involved with the immune system
62
GAL4-UAS System
Enhancer sequences in yeast regulating transcription of genes in galactose metabolism
parallels lac operon
When galactose is the only sugar available, yeast induce transcription of four
genes that import and break down galactose
63
GAL genes
* 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
64
GAL; absence of galactose
Gal 4 is bound by Gal80 and is unable to activate transcription
@ activation domain UASG
65
GAL; galactose present
Galactose and GAL3 protein bind to GAL80, freeing GAL4
activates transcription
66
GAL used in the fly genome
Widely used in Drosophila transgenic lines as an “ON/OFF” switch for genes
* 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
67
Insulator sequences
located between enhancers and promoters of genes
They can block enhancer activity towards a specific promoter and redirect it to another gene
68
Insulator and enhancer interactions
Insulators are thought to block expression of
promoters by directing the formation of loops in DNA through regulatory proteins
loops contain enhancers and the genes they activate
69
Chromosome 11 - differential genomic imprinting
IGF2 and H19
| H for her
F for father
Maternal chromosome
- enhancer drives expression of H19
- insulator blocks IGF2 expression
Paternal chromosome
- methylation inactivates the ICR (insulator)
- blocks H19 expression
- enhancer drives IGF2 expression
70
Heterochromatin
can silence gene expression
71
Euchromatin
maintains activated gene expression
72
Chromatin
Unwound dna
structure regulates gene expression
can be remodeled by enzymes that alter associations with DNA and nucleosomes
ex. methylation, demethylation, acetylation, deacetylation
73
RNA interference (RNAi)
Post-transcriptional regulation of gene expression
* Double stranded RNA gets processed by enzyme Dicer into 21-24 bp fragments
* 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
* The guide strand complementary base pairs with mRNA, destroying it before translation, silencing
gene expression
74
microRNA (miRNA)
dsRNA expressed within a cell
75
Short interfering RNA (siRNA)
dsRNA from external
sources (e.g. virus)
76
The guide RNA bound to RISC directs one of three gene-silencing processes
1. RISC guide RNA binds to mRNA through
complementary base pairing and destroys it
2. RISC guide RNA binds mRNA and prevents
translation
3. RISC complex directs chromatin modifying
enzymes to the nucleus where transcription
of selected genes are silenced
77
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
78
Dnase I
hypersensitive sites
Regions that are sensitive to Dnase I digestion
* These regions contain euchromatin and are transcriptionally
active
* A method of detecting transcriptionally active areas in a genome
79
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
80
4 examples of regulated gene expression
1. Embryonic development
2. Caste changes in eusocial insects
3. Stress response
4. Circadian clock
and more!
81
Embryonic development (regulated gene expression)
- various genes and enhancers coordinate the body plans of embryos
- location within the embryo correlates to larvae location and then location of the fly's body
82
Royal jelly and queen honey bees (regulated gene expression)
* 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”
83
Stress response (regulated gene expression)
* 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
84
Circadian clock (regulated gene expression)
* 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
