1
Q
gene regulation
A
- cell can control level of gene expression
- structural genes are regulated- so proteins produced at certain times and in specific amounts
- “constitutive” genes are unregulated
- constant levels of expression
- “housekeeping genes”
2
Q
benefits of gene regulation
A
- conserves energy
- genes expressed in appropriate cell type and at the correct stage in development
- results in cell differentiation
- same genome, different proteomes
- ex: skeletal muscle cell, neuron and skin cells
3
Q
Regulation through development
A
- globin consists of 4 polypeptides
- composition of hemoglobin protein changes during development
- fertilization; embryo-> fetus -> adult
- fetal hemoglobin- higher oxygen affinity
4
Q
Hoxc8 example
A
- backbone development
- nearly identical in several vertebrates
- but, chicken has 7 vertebrae, mouse has 13
- why the diversity?
- transcribed more in mouse
5
Q
Gene regulation can occur at different points; prokaryotes
A
- transcription level regulation
- common in bacteria
- controls how much mRNA is made
- efficient - control rate of translation
- regulate at protein or post-translation
6
Q
gene regulation at different points, eukaryotes
A
- 4 methods
1. transcriptional regulation (common)
2. RNA processing - splicing
- degradation rates
3. translation
4. post translation - feedback inhibition
- protein modifications
- degration rates
7
Q
transcriptional regulation in bacteria
A
- involves regulatory transcription factors
- bind near promoter, affect trasncription of one or more nearby genes - repressors inhibit transcription
- negative control - activators increase the rate of transcription
- positive control - involves small effector molecules
8
Q
Small effector molecule ; transcriptional regulation in bacteria
A
- binds to transcription factor-> conformational change
- no effector-> repressor bound to DNA-> gene off
- effector-> repressor not bound to DNA-> gene on
- alters transcription factors ability to bind to DNA
- 2 domains in transcription factor that respond to small effector molecules
- site where protein binds to DNA
- site for small effector molecule binding
9
Q
Operons
A
- cluster of genes controlled by one promoter
- only in prokaryotes
- genes transcribed together on a single mRNA
- Polycistronic: different genes ONE mRNA moelcule
- allows efficient regulation of a group of genes with a common function
10
Q
what would be the action of a transcription factor that acts an activator?
A
-increase in the number of mRNAs transcribed
11
Q
lacP
A
- promoter
- drives expression of multiple genes
12
Q
lacZ
A
-B-galactosidase: enzyme for catabolizing lactose
13
Q
lacY
A
lactose permease: enzyme for transporting lactose
14
Q
lacA
A
galactoside transacetylase- function unknown
15
Q
near lacP, 2 regulatory sites
A
- lacO- operator- provides binding site for repressor protein
- CAP site- activator protein binding site
16
Q
lacl gene
A
- codes for lac repressor
- considered a regulatory gene since its sole function is to regulate other genes expression
- has its own promoter (not part of lac operon)
17
Q
lac operon: lactose present
A
- allolactose binds to lac repressor; inactivates repressor
- process called induction and lac operon is inducible
18
Q
lac operon; lactose absent
A
- Lac repressor binds to operator site; prevents RNA polymerase from transcribing lacZ, lacY and lacA
- RNA polymerase can bind but not move forward
19
Q
lac operon; glucose present
A
- presence of glucose represses the lac operon
- known as catabolite repression: when the presence of another energy source represses expression of another gene
- cAMP is a small effector moelcule present ONLY when glucose is absent
- cAMO binds to an activator protein called catabolite activator protein (CAP)
- bacteria cannot express lac operon efficiently without CAP
- high glucose= no cAMP= inactive CAP= no lac transcription
- hence, no processing of lactose
20
Q
lac operon; glucose low
A
- cAMP is high
- cAMP binds/ activates to CAP
- activated CAP binds to CAP site near lac promoter
- resulting bend in DNA enhances RNA polymerase binding which increases transcription
21
Q
lac operon; both lactose and glucose high
A
- lac operon is shut off by low cAMP
- glucose uptake causes cAMP levels to drop
- CAP does not activate transcription
- bacterium uses one sugar at a time, glucose
22
Q
lac operon; lactose is high and glucose is low
A
- lac operon is turned on
- allolactose levels rise and prevent lac repressor from binding to operator
- CAP-cAMP complex is bound to the CAP site
- bacterium uses lactose
23
Q
transcription regulation in eukaryotes
A
- follows some of the same principles found in prokaryotes
- presence of activator and repressor transcription factors (TFs)
- many TFs regulated by small effector molecules
- many important differences
- genes almost always organized individually; no operons!
- more highly regulated -> increased complexity
24
Q
activators
A
activator proteins stimulate RNA polymerase to initiate transcription
25
repressors
repressor proteins inhibit RNA polymerase from initiating transcript
26
modulation (controls gene expression)
small effector molecules, protein-protein interactions, and covalent modifications can modulate activators and repressors
27
chromatin (control gene expression)
activator proteins promote loosening up of the region in the chromosome where a gene is located, making it easier for RNA polymerase to transcribe the gene
28
DNA methylation (control gene expression)
usually inhibits transcription, either by blocking an activator protein or by recruiting proteins that make DNA more compact
29
combinatorial control of gene expression
most mechanisms are aimed at controlling the initiation of transcription at the gene promoter
30
3 features in eukaryotic promoters; 1. TATA box
1. TATA box- 25 base pairs upstream from transcriptional start site
31
3 features in eukaryotic promoters; 2. Transcriptional start site
- where transcription begins, with TATA box forms core promoter
- core promoter alone results in low level basal transcription
32
3 features in eukaryotic promoters; 3. Response elements
- enhancer and silencer transcription factors
- binding of TFs regulate transcription at the core promoter
- usually 50-100bps upstream; can be 100,000bps away
33
3 protein complexes needed for transcription
1. RNA polymerase II
2. general transcription factors
- GTFs + RNA Pol II come together at core promoter before transcription initiation
- pre initiation complex- assembled GTFs and RNA Pol II at the TATA box; form basal transcription apparatus
3. mediator- composed of several proteins
- partially wraps around GTFs and RNA Pol II
- mediates interactions with activators or repressors
- controls rate at which RNA Pol II can begin transcription
34
response elements control of transcription
- activators bind to enhancer sequences
- repressors bind to silencer sequences
- regulate rate of transcription of a nearby gene
- most TFs do not bind directly to RNA Pol II
35
3 ways to control RNA polymerase II
1. By promoting the assembly of the pre initiation complex
- results in a basal rate of transcription
2. control rate of RNA Pol II transcription via mediator
- activators "turn-on" mediator-> increased transcription
- repressors "turn-off" mediator -> decreased transcription
3. recruit proteins that influence DNA packaging
- DNA is NOT naked
- proteins bind and compact DNA into chromatin
- packaging affects gene expression
- whether RNA Pol can "get" to a gene
- heterochromatin vs. euchromatin
36
unpacking DNA
- some activators "unwind" DNA near a gene
- recruit proteins to loosen DNA compaction
- histone acteyltransferase (HAT) attaches acetyl groups to histone proteins so they don't bind DNA as tightly
37
DNA methylation- turning genes off
- DNA methylase attaches methyl groups to DNA
- usually inhibits transcription
- common in some eukaryotes but not all
- generally occurs at "CpG islands"
- region of high C and G phosphodiester bonds
- near promoters in vertebrates and plants
- methylated CpG islands are correlated with repressed genes
38
Methylation inhibits transcription in 2 ways
1. methylation of CpG islands prevents activator binding
| 2. converts chromatin fro open to closed
39
regulation of RNA processing and translation in eukaryotes
- unlike bacteria, gene expression commonly regulated by RNA processing
- added benefits include
- produce more than one mRNA transcript from a single gene (gene encodes 2 or more polypeptides)
- more complex proteome can be made
- faster regulation achieved by controlling steps after mRNA transcript made
- alternative splicing
40
alternative splicing of pre-mRNAs
- causes mRNAs to contain different patterns of exons
- alternative proteins generally have similar functions
- allows same gene to make different proteins
- at different stages of development
- in different cells types
- in response to a change in the environmental conditions
41
splicing across evolutionary history
frequency of alternative splicing increases with increasing biological complexity
42
translational regulation- miRNAs
- microRNAs (miRNAs)- AKA RNA interference (RNAi)
- small RNA molecules that silence translation of mRNAs
- degrade mRNA or block translation
- common in animals and plants
43
translational regulation: iron binding example
- iron= cofactor for many enzymes
- but toxic at high levels
- mammalian cells make protein ferritin
- forms a hollow, spherical complex to store excess iron
- translation of the mRNA that codes for ferritin is controlled by an RNA-binding protein known as the iron regulatory protein (IRP)
44
different levels of iron
- when iron levels in the cytosol are low, ferritin is not needed
- IRP binds to a response element within the ferritin mRNA
- inhibits translation
- when iron is abundant, iron binds to IRP and prevents it from blocking translation
- ferritin mRNA is translated to make more ferritin protein
- faster than transcriptional regulation, which requires activation, transcription, and translation of ferritin gene
BIO 111
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