1
Q
negative regulation
A
- when bound to DNA, repressor protein prevents transcription
- competition between RNA polymerase and repressor protein for promoter binding
2
Q
positive regulation
A
- when bound to DNA, activator protein promotes transcription
- activator protein recruits RNA polymerase to the promoter to activate transcription
3
Q
two types of way in which ligands can affect transcription in prokaryotes
A
- ligand binds to remove regulatory protein from DNA:
- addition of ligand switches gene on by removing repressor protein
- addition of ligand switches gene off by removing activator protein - ligand binds to allow regulatory protein to bind to DNA:
- removal of ligand switches gene on by removing repressor protein
- removal of ligand switches gene off by removing activator protein
4
Q
Lac repressor
A
negative regulation where addition of ligand switches gene on by removing repressor protein
5
Q
Trp repressor
A
negative regulation where removal of ligand switches gene on by removing repressor protein
6
Q
catabolite activator protein
A
positive regulation where removal of ligand switches gene off by removing activator protein
7
Q
many of the proteins that bind to DNA have a
A
helix-turn-helix
8
Q
where are gene regulatory elements typically found?
A
- typically close to the transcriptional start site of prokaryotic genes
- but, can also be found far upstream of the gene (really negative), downstream of gene (after; more common in eukaryotes) and within the gene (introns; only in eukaryotes)
9
Q
give an example of how can regulatory elements that are distant from the transcriptional start site influence transcription?
A
- NtrC protein is a transcriptional activator
- DNA looping allows NtrC to directly interact with RNA polymerase to activate transcription from a distance
10
Q
bacteriophage lambda
A
- virus that infects bacterial cells
- attaches to host cell and injects lambda DNA, which then circularises
- positive and negative regulatory mechanisms work together to regulate the lifestyles of bacteriophage lambda
11
Q
bacteriophage lambda can exist as one of two states in bacteria
A
- prophage pathway: happens under favourable bacterial growth conditions
- lytic pathway: happens when host cell is damaged
12
Q
prophage pathway
A
- integration of lambda DNA into host chromosome
- under favourable conditions, cell division will take place: DNA polymerase will also replicate the viral DNA that has been incorporated
- integrated lambda DNA replicates along with host chromosome
13
Q
lytic pathway
A
(- induction event)
- synthesis of viral proteins needed for formation of new viruses
- rapid replication of lambda DNA and its packaging into complete viruses
- cell lysis releases a large number of new viruses
14
Q
what proteins are responsible for initiating the switch between prophage and lytic pathways?
A
- lambda repressor protein (cI)
- Cro protein
they repress each other’s synthesis, giving rise to the two states
15
Q
what direction is RNA synthesised in?
A
5’ to 3’
16
Q
what type of genetic information does bacteriophage lambda have?
A
double stranded DNA
17
Q
stable state 1: the prophage state
A
- lambda repressor occupies the operator
- blocks the synthesis of Cro
- activates its own synthesis
- most bacteriophage DNA is not transcribed
18
Q
stable state 2: the lytic state
A
- Cro occupies the operator
- blocks synthesis of lambda repressor
- allows its own synthesis
- most bacteriophage DNA is extensively transcribed
- DNA is replicated, packaged, new bacteriophage is released by host cell lysis
19
Q
is Cro an activator?
A
NO; it ALLOWS its own synthesis, but, unlike lambda repressor, doesn’t activate it by recruiting RNA polymerase
20
Q
what triggers the switch between prophage and lytic states?
A
the prophage-lytic control is an example of a transcriptional circuit:
- host response to DNA damage causes switch to lytic state, inactivating repressor
- under good growth conditions, lambda repressor protein turns off Cro and activates itself in a positive feedback loop, maintaining prophage state
21
Q
4 types of transcriptional circuits + draw them
A
- positive feedback loop (eg lambda repressor protein)
- negative feedback loop
- flip-flop device (indirect positive feedback loop) - eg Cro/Repressor switch
- feed-forward loop
22
Q
positive feedback loop
A
can be used to create cell memory:
1. in the parent cell, transcription regulator A is not made because it is normally required for the transcription of its own gene
2. there is an initial transient signal which turns on the expression of gene A
3. protein A causes increased transcription of gene A, causing gene A to continue to be transcribed in absence of initial signal in progeny cells
23
Q
feed-forward loop circuit
A
- can measure the duration of a signal
- both A and B are required for transcription of Z - this decreases the sensitivity (i.e. you need a lot more input to get the output)
brief input: B does not accumulate and gene Z is not transcribed
prolonged input: B accumulates and gene Z is transcribed
24
Q
why is understanding of transcriptional circuits so essential?
A
- combinations of regulatory circuits combine in eukaryotic cells to create exceedingly complex regulatory networks
- scientists can construct artificial circuits and examine their behaviour in cells: this is called synthetic biology
25
the Repressilator
- an example of synthetic biology
- scientists created a simple gene oscillator using a delayed negative feedback circuit
- this is known as a proof of principle experiment
26
draw the Repressilator and explain how it works
1. A expressed
2. B repressed
3. C expressed
4. C represses A expression
5. A repressed
6. B expressed
7. C repressed
8. repeat 1-4
27
predicted vs observed results of the repressilator
predicted: regular up and down for each protein
observed: increasing amplitude of fluorescence (amount of protein) due to bacterial growth
28
transcriptional attenuation
a premature termination of transcription that happens in both prokaryotes and eukaryotes
29
give 2 examples of how transcriptional attenuation can be achieved
- RNA adopts a structure that interferes with RNA polymerase
- regulatory proteins can bind to RNA and interfere with attenuation
30
riboswitches
- used by prokaryotes, plants, and some fungi to regulate gene expression
- short RNA sequences that change conformation when bound by a small molecule
31
example of a riboswitch
prokaryotic riboswitch that regulates purine biosynthesis
low guanine levels:
- transcription of purine biosynthetic genes is on
high guanine levels:
- guanine binds riboswitch
- riboswitch undergoes conformational change
- causes RNA polymerase to terminate transcription
- transcription of purine biosynthetic genes is off
32
pyrimidines vs purines
pyrimidines: one ring (C, T, U)
purines: two rings (A, G)
33
distinguish between RNA transcription in prokaryotes and eukaryotes
- in eukaryotes, different RNAs are transcribed by different RNA polymerases
- in prokaryotes, there is a single type of RNA polymerase
34
RNA polymerase I
rRNA genes
35
RNA polymerase II
- transcribes all protein-coding genes
- requires 5 general transcription factors: TFIID, TFIIB, TFIIF, TFIIE and TFIIH (prokaryotes only need one; sigma factor)
36
RNA polymerase III
tRNA genes
37
eukaryotic transcription
- transcription initiation in eukaryotes requires many proteins named general transcription factors
- these help position RNA polymerase at eukaryotic promoters
38
transcription factor nomenclature
TF = transcription factor
I/II/III = type of RNA polymerase
D = letter
39
TATA box
- for RNA polymerase II, many eukaryotic promoters contain a TATA box
- promoters may have other consensus sequences
40
TBP
TATA Binding Protein - a subunit of TFIID that recognises TATA box and other DNA sequences near the transcription start point
41
TFIIH
unwinds DNA at the transcription start point, phosphorylates Ser5 of the RNA polymerase C-terminal domain (CTD); releases RNA polymerase form the promoter
42
3 features of eukaryotic transcription
- eukaryotic genomes lack operons
- eukaryotic DNA is packaged into chromatin which provides an additional mode of regulation
- eukaryotic transcriptional activation requires many gene regulatory proteins
43
2 categories of eukaryotic transcription factors
general transcription factors
gene regulatory proteins.
44
2 categories of gene regulatory proteins
activators and repressors
45
mediator
acts as an intermediate between regulatory proteins and RNA polymerase (optional)
46
gene regulatory proteins
- many (~2000 encoded by the human genome)
- both activators and repressors
- can act over very large distances, sometimes >10,000 base pairs away
- one mechanism is DNA looping
- often function as protein complexes on DNA
47
coactivators and corepressors
assemble on DNA-bound gene regulatory proteins - do not directly bind to DNA
48
structure of eukaryotic activator proteins
have a modular design:
1. DNA binding domain (DBD): recognises specific DNA sequence
2. activation domain (AD): accelerates frequency/rate of transcription
in nature (with mutations) or in biotechnology, you can mix ad match DBDs and ADs.
49
how do activator proteins activate transcription?
attract, position, and modify:
- general transcription factors
- mediator
- RNA polymerase II
they can do this either:
1. directly by acting on these components: can bind directly to transcriptional machinery or the mediator and attract them to promoters (like prokaryotic activators)
2. indirectly modifying chromatin structure and increasing promoter accessibility
50
nucleosomes
basic structure of eukaryotic chromatin
- DNA wound around a histone octamer
- [H2A, H2B, H3, and H4] x 2
- DNA wraps around the core histones 1.66667 times
51
linker DNA
10-80bp long
52
DNA wrapped around histone
~147bp long
53
H1
paperclip holding DNA to histones
54
2 models of nucleosome models and their significance
zigzag model
solenoid model
some evidence for both, but, either way, transcriptional machinery cannot assemble on promoters tightly packed in chromatin
55
4 major ways in which activator proteins can alter chromatin
- nucleosome sliding
- histone removal
- histone replacement
- specific pattern of histone modification
56
nucleosome sliding
nucleosome structure can be altered by chromatin remodelling complexes in an ATP-dependent manner to increase promoter accessibility
57
histone removal and replacement
nucleosome removal and histone exchange requires cooperation with histone chaperones and ATP-dependent chromatin-remodelling complexes
58
specific pattern of histone modification
- histone modifying enzymes produce specific patterns of histone modifications known as 'histone code'
- histone modifications occur on specific amino acids of histone tails
- phosphorylation, acetylation, methylation
59
phosphorylation
- addition of a phosphate group
- enzyme: kinase
60
acetylation
- addition of acetyl group
- enzyme: acetyltransferase
61
methylation
- addition of methyl group
- enzyme: methyltransferase
62
nickname for histone modifying enzymes
'writers'
63
what tends to be the composition of the histone tails?
more commonly the N terminal portion of the histone proteins; however, there are some C terminal tails too
64
how is the histone code interpreted?
code 'reader' proteins can recognise specific modifications and provide meaning to the code
65
give an example of a histone code
Human Interferon Gene Regulation:
1. activator protein binds to chromatin DNA and attracts a histone acetyltransferase (HAT)
2. HA acetylates lysine 9 of histone H3 (H3K9) and lysine 8 of histone H4 (H4K8)
3. Activator protein attracts a histone kinase (HK)
4. histone kinase phosphorylates serine 10 of histone H3 (H3S10). can only occur after acetylation of lysine 9.
5. serine modification signals the acetyltransferase to acetylate lysine 14 of histone H3.
the histone code for transcription initiation is written.
6. TFIID and a chromatin remodelling complex bind to modified histone tails and initiate transcription
NB: you cannot skip steps, you must proceed in this order. also, this is ONLY EUKARYOTES.
66
how does transcriptional repression in eukaryotes compare to that in prokaryotes?
- unlike prokaryotes, eukaryotic repressor proteins rarely compete with RNA polymerase for access to DNA
- instead, they use a variety of mechanisms to inhibit transcription
67
structure of repressor proteins
DNA binding domain and repression domain (RD)
68
5 mechanisms of transcriptional repression in eukaryotes
1. competitive DNA binding by repressor
2. masking the activation surface (activation domain)
3. direct interaction with the general transcription factors
4. recruitment of chromatin remodelling complexes to make chromatin more compact
5. recruitment of histone deacetylases
6. recruitment of histone methyl transferases
-> 5 + 6 alter the histone code for repression
69
spreading of histone code
- carried out by reader-writer complexes
- reader binds an existing histone mark and recruits a writer
- writer adds a reader to the next nucleosome
- positive feedback copies the mark along chromatin
70
how is methylation involved in the spreading of histone code?
- DNA methylase enzyme is attracted by reader and methylates nearby cytosines in DNA
- DNA methyl binding proteins bind methyl groups and stabilise structure
- methylation and therefore gene expression patterns can be inherited (process called epigenetic inheritance)
71
define epigenetics
heritable changes in gene expression caused by mechanisms other than changes in the underlying DNA sequence
BIO230
flashcards
Decks in class (26)
# Cards
-
Lecture 163
-
Lecture 271
-
Lecture 368
-
Lecture 4.47
-
Lecture 555
-
Lecture 659
-
Lecture 1 - Membrane Trafficking45
-
Lecture 2 - Cytoskeletal Networks58
-
Lecture 3 - Cell Adhesion54
-
Lecture 4 - Tissue Morphogenesis50
-
Lecture 5 - Tissue Patterning50
-
Lecture 6 - Tissue Renewal & Stem Cells55
-
Lecture 7 - Concepts in Cell Signaling69
-
Lecture 8 - Signaling via Small Molecules40
-
Lecture 9 - Signaling via Protein Modifications41
-
Lecture 10 - The Cell Cycle40
-
Lecture 11 - Programmed Cell Death43
-
Lecture 12 - Cancer59
-
L1-L3: How are cells and tissues organised spatially?157
-
L4-L6: How do multicellular organisms develop?155
-
L7-L9: How do cells communicate with each other?150
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l840
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l941
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l1040
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l1143
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1259
