REGULATION OF GENE EXPRESSION Flashcards
(127 cards)
1
Q
What is the transcription of many genes controlled by?
A
Activators and repressors
2
Q
What is the activity of activators and repressors controlled by?
A
Inducer or codepressors
3
Q
Why are all the genes not expressed at once?
A
In prokaryotes- different genes are expressed in response to different environments e.g. food present such as lactose
- Also don’t waste energy expressing genes which don’t need to be used
4
Q
What do eukaryotic genes need to express?
A
DIFFERENT FACTORS as they need to be able to differentiate into different cell types
5
Q
What is the ground state for bacterial genes?
A
They are always ON unless inactivated
6
Q
What is the ground state for eukaryotic genes?
A
They are always OFF unless activated
- They need another 10-20 proteins to activate RNA polymerase transcription
7
Q
Why are eukaryotic genes hard to access?
A
- Because they are wrapped tight in chromosomes (histone)
8
Q
What does regulation at the transcription level do?
A
- Regulate how much mRNA is made from a gene
- Without mRNA final protein can’t be expressed
- Most common level of regulation
9
Q
What does regulation at the level of translation do?
A
- Regulates how much active protein is made from mRNA transcript
10
Q
What determines where RNA polymerase starts transcription?
A
- Promoter sequences e.g. TATAAT or TTGACA
11
Q
What is negative regulation?
A
- When a regulatory protein is used to stop transcription of a gene(turns promoter OFF)
- Repressor
12
Q
What is the RNA polymerase in bacteria recruited to the promoter by?
A
- By sigma factor
13
Q
When does negative regulator need to be inactivated?
A
- When genes need to be turned on in response to a stimulus
14
Q
What is positive regulation?
A
- Activator
- Regulatory protein required before the gene is efficiently transcribed (turn promoter ON)
15
Q
When does a positive promoter need to be activated?
A
- When genes need to be turned on in response to a stimulus
16
Q
What will slightly different promoter sequences be recognised by/how is RNA polynmerase (prok) and thounsands of genes explained?
A
- Different sigma factors recognising alternative promoter sequences
e. g sigma 32 (genes induced by heat shock) so levels of this then rise when heat shock happens
17
Q
What is the place where the repressor binds called?
A
- The Operator site
- (promoter and operator overlap)
18
Q
How do repressor proteins stop transcription?
A
- By repressor binding over the promoter sequence so RNA polymerase can’t bind to the DNA
19
Q
What is sometimes required for the repressor to bind(negative regulation of transcription)?
A
- A corepressor
20
Q
Repressor + corepressor=
A
ACTIVE site for binding
21
Q
What is the region between -35 and +1?
A
- The promoter region (overlaps with the operator region)
22
Q
What section on the DNA is the operator region?
A
-5 to +21
23
Q
What happens to the repressor in negative regulation to allow for transcription?
A
It binds to the inducer (no longer binding to the operator) to allow for transcription to occur
24
Q
How is repression relieved?
A
By an inducer
25
What are the three steps in allowing transcription in negative regulation?
1. Repressor binds to the operator site and stops transcription
2. When inducer is present it binds to repressor and repressor falls off DNA
3. RNA polyemerase can now access promoter and direct transcription
26
What is the repressor and inducer for lactose respectively?
- LacI (repressor) and allo-lactose (inducer)
27
What occurs in positive regulation of transcription?
Activator can only bind to DNA in presence of inducer (inducer interacts with the activator allowing it to bind to DNA)
28
What are the two steps in positive gene expression to allow for activation?
1. Activator must bind to promoter to allow transcription to begin
2. Activator can only bind to DNA in presence of inducer
29
Where is the binding site for activator proteins?
- Upstream of the promoter
- needs inducer to bind
e. .g maltose is an inducer (binds to activator protein--> changes conformation--> can then bind to activator site upstream of promoter.)
30
What are allosteric proteins?
Proteins that exist in two different conformational forms (shapes)
- Change conformation when binding to the inducer
31
What are examples of allosteric proteins?
- Transcriptional repressors and activators
32
What does the change in conformation of allosteric proteins alter?
- The ability to bind DNA
33
What are the two different binding sites that the allosteric proteins have?
- One for binding inducer
| - One for binding DNA
34
What is the negative regulation of the Lac operon controlled by?
- Lactose repressor (product of LacI)
| - Inducers are low MW beta-galactosides (allo-lactose)
35
What is the positive regulation of the lac operon controlled by?
- CAP protein
| - Inducer is cAMP ( which responds to glucose)
36
What does the lac operon sense?
- not the actual glucose concentration BUT the cAMP levels
| - Levels of cAMP and positive regulator CAP (catabolite activator protein)- catabolite repression
37
What do glucose levels regulate?
cAMP levels
38
Will the lac operon be expressed when glucose is present?
NO!!! It won't because glucose has first preference
39
What does catabolite repression allow for?
- The fast adaptation of bacteria to a preffered carbon source
40
What happens in levels of high glucose?
- No cAMP produced
41
What happens in levels of low glucose?
- cAMP produced from ATP
42
How does the cAMP-CAP complex activate transcription in positive regulation?
- Complex binds to promoter and activates transcription
| -
43
What effect does CAP have on DNA?
- It 'bends' the DNA (tortional pressure separates the DNA)
44
What does bent DNA allow for in positive regulation?
For RNA polymerase to access the promoter more efficiently
45
When does CAP activate transcription efficiently?
- In the absence of the lac repressor
46
When is there high levels of the lac operon expression?
- When glucose is absent or lactose is present
47
What happens when there is glucose present (cAMP low) and no lactose?
- Repressor binds (CAP doesn't bind)
| - NO EXPRESSION OF LAC mRNA
48
What happens when glucose is present (cAMP low), and lactose present?
- Repressor doesn't bind, CAP doesn't bind
| - LITTLE EXPRESSION OF LAC mRNA
49
What happens when glucose is absent (high cAMP) and lactose is present?
- The CAP binds (CAP cAMP complex)
| - HIGH EXPRESSION OF LAC mRNA
50
The lac operon has both:
- Positive and negative regulation (Lac repressor and CAP activator)
51
Which example of regulation is the lac operon?
- Regulation (both positive and negative) of INDUCIBLE genes
52
What are inducible genes?
- Those which are expressed in response to an environmental change (food) or dependent on position in cell cycle
53
Why are some genes inducible whilst others are repressible?
- Depends which side of metabolism they are on
e.g. whether the genes are on the destructive path of metabolism (breaking down-catabolism)
or constructive path (anabolism- enzyme building--> biosynthesis)
54
What is a repressible gene?
- If function of genes product is to synthesise a molecule, and molecule is present in abundance, molecule will serve as a CO-REPRESSOR of that repressible gene--> switches gene OFF e.g. trp operon
55
How are anabolic (biosynthetic) pathways regulated?
- Feedback repression (gene expression)
| - This is negative control of gene expression
56
What are the two ways of negative control of gene expression?
- Repression of transcription initiation
| - Inducing premature termination of transcription (attenuation)
57
What is attenutation?
- Inducing premature termination of transcription
58
When we say 'inhibition' what do we always refer to?
- Protein activity
59
How many enzymes are needed to synhesise Tryptophan?
5
60
What is the definition of FEEDBACK INHIBITION?
" Where the product of a pathway inhibits the ACTIVITY of an ENZYME earlier in the pathway
- often the end product in the pathway will bind to an enzyme for the first step reducing its activity.
61
What is feedback REPRESSION?
" Where the product of the pathway INTERACTS with regulatory protein (e.g. repressor) to stop TRANSCRIPTION OF GENES encoding enzymes in pathway. e.g. lac operon"
62
What occurs in low trp levels?
- involves action of allosteric repressor protein
- Repressor cannot normally bind to operator site on DNA
- So NO REPRESSION OF TRANSCRIPTION
(meaning that transcription of the entire trp operon occurs)
63
What occurs in high trp levels?
- Repressor can bind to the molecule that is the end product of the pathway (e.g. tryptophan amino acid)
- changes conformation
- can bind to operator site (high affinity for DNA) on DNA
- REPRESSES TRANSCRIPTION
64
Is the gene for the repressor part of the operon it controls?
NO!
65
What is common to the LacI and TrpI repressors?
- BOTH bind to the operator site DOWNSTREAM from the promoter
- BOTH perform same role- block RNA polymerase from transcribing genes
66
What is the difference between the LacI and trpI repressors?
- Differs in how the repressor interacts with its effector molecule
- e.g. in lac operon,- in absence of effector, molecule repressor is ACTIVE (bind to operator and block transmission)
Trp operon--> without effector, repressor not active (only in presence of trypotphan (corepressor) is it able to bind to DNA and BLOCK transcription
67
What is the trp operon regulated by?
- The TrpR repressor
| - Also has a second level of control (transcriptional attenuation)
68
What does transcriptional attenuation in trp operon involve?
- An upstream leader peptide (L) that is tryptophan rich
69
What does the TrpL sequence have?
- Trp-Trp (two tryptophans in sequence)-two codons
70
What is transcribed when tryptophan is present?
Only the leader sequence and then transcription stops
71
What is transcribed when tryptophan is absent?
The whole mRNA is transcribed
72
What occurs in transcriptional attenuation?
- Turns off Trp operon when not needed
- Trp operon is transcribed as a single polycistronic mRNA
- Leader sequence is LONG (two codons -Trp-Trp)
73
What is transcriptional attenuation in more detail?
- There are 4 regions that can form stem and loop structures (hydrogen bonding)
- called segments 1,2,3,4
- They can form 1:2 2:3 3:4 loop structures
74
Which segment is the UGGUGG that encodes the two Trp segments?
- In segment 1
75
What occurs when there is a HIGH tryptophan level in terms of attenuation?
- As soon as segments 1 &2 form, ribosome binds
| - Only segments 3&4 form the IDEAL terminator becasue of the UUUUUUU
76
Which segments form the ideal terminator?
- Segments 3&4
77
What occurs in a low trp level in terms of attenuation?
- Ribosome comes but because low levels of Trp, there is no tRNA (rate limiting) so the ENITRE Trp operon is transcribed
78
Do prokaryotes have post transcriptional modifications?
No they don't have any real post transcriptional modifications
79
What post transcriptional modification occurs in eukaryotes?
- Addition of 7-methylguanine cap at 5' end
- Addition of poly A tail
- Splicing to remove introns
80
What is the function of the 5' cap addition?
- Protects hydrolysis by exonucleases
- Tells the cell the 5' end is intact
- Functions as an "attach here" signal for ribosomes
- Modified methyl guanosine is added 5'-5'
81
What does the modified methyl guanosine added 5'-5' do?
- Confuses exonucleases that degrade mRNA from 3' end
82
What is the function of the 3' tail?
- Increased stability of mRNA (protection from exonucleases)
- Has a role in the export out of nucleus (motility)
- Essential for initiating translation of some mRNAs
- Adds about 200 As
83
What sequence is recognised to do a poly A tail (3')?
AAUAAA
84
What is the poly A tail catalysed by?
- Poly (A) polymerase (PAP) and involves CPSF protein (cleavage and polyadenylation specificity factor)
85
How many types of RNA polymerases do eukaryotic cells have compared with prokaryotes?
- Eukaryotes have 3 types whereas prokaryotes only have 1 type
86
What does RNA polyemerase I do?
- Transcribes rRNA genes
87
What does RNA polymerase II do?
- Main one
| - Produces mRNA and transcribes some snRNA genes and miRNAs
88
What does RNA polymerase III do?
Transcribes most small RNA genes (tRNA, some snRNAs 5S rRNA)
89
What is the rough process of transcription initiation in prokaryotes?
- RNA polymerase recognising and binding directly to the promoter region via sigma factor subunit -35 and -10 regions in E coli promoters
90
What is the process of transcription initiation occurring in eukaryotes?
- transcription factors binding to promoter (TATA box) and promoter proximal elements
- Recognition involves TATA box (25-30 bp upstream from transcription START site)
1. Binding of TATA box binding protein (part of TFIID)
2. Binding causes bending of DNA -landmark to attract other general transcription factors (same for all eukaryotic genes)
91
Which particular part of RNA polymerase II in eukaryotes plays an important role in coordinating all 3 processing events (5' capping, 3' poly A tail and splicing) ?
- CTD: Carboxy Tail Domain of RNA polymerase II
92
When making a protein, which end comes out first N or C terminus?
The N terminus first then C terminus last
93
How do neurons find their target?
They extend a growth cone at end of axon
94
What (in genera) controls axon guidance?
Environmental signals
| e.g. attraction/repulsion, guidance cues
95
What can attractive and repulsive signals cause respectively?
- Attractive directs actin fibres to extend by encouraging polymerisation and repulsive signals cause actin filaments to retract (depolymerise)
96
What are microRNAs?
- Produced in nucleus and receive 5' cap and 3' tail
- Have hairpin with one mismatch
- While microRNA is in nucleus it gets cropped and loses 5' cap and 3' tail
97
What is the difference in transcription and translation in prokaryotes and eukaryotes?
- Transcription and translation occur at the same time in CYTOPLASM , as soon as 5' end comes out, ribosomes attach (PROKARYOTES)
EUK: Transcription occurs in nucleus, and translation in cytoplasm
- Processes NOT CONCURRENT
98
What is the difference between pro and euk for post transcriptional modification?
PRO: No real post TRANSCRIPTIONAL modification
EUK: Synthesised as pre-mRNA
- Pre mRNA processed as it is transcribed (addition of 7-metylguaninie cap at 5' end, addition of Poly A tail, splicing to remove introns)
99
What is the function of the 5' cap in euk?
- Protects from hydrolysis by exonucleases
- Tells the cell that 5' end is intact
- Functions as 'attach here' signal for ribosomes
- Modified methyl guanosine is added 5'-5' (confuses the exonucleases that degrade mRNA from 3' end)
100
What is the function of the 3' Poly A tail in Euk? What is it catalysed by and what protein is involved?
Increased stability of mRNA (protection from exonucleases)
- Has a role in export out of nucleus
- Essential for initiating translation of some mRNAs
- Adds about 200 As
- Catalysed by Poly (A) polymerase (PAP) and involves CPSF protein (cleavage and polyandenylation specificity factor)
Recognises AAUAAA to do poly A tail
101
When trascription begins in euk, what do the CPSF protein do to help form the Poly A tail?
- After transcription begins, CPSF recruited to CDT(PPPPPP) and rides on it until AAUAA sequence transcribed
- CPSF binds to AAUAA sequence instead
- Additional cleavage factors recruited to cleave RNA from polymerase
- Polymerase PAP adds 200 adeylate residues to 3' end of transcript
102
What do PAP and CPSF stand for ?
- Poly (A) Polymerase
| - Cleavage and Polyadenylation Specificity Factor
103
What is the stability like in pro and euk in terms of degradation in cell?
PROK: mRNA has triphosphate group and is susceptible to degradation from exonucleases (so will remain UNSTABLE IN CELL and be degraded after a minute)
EUK: Protected by 5' cap and poly A tail to protect from exonuclease degradation (only endcodes one protein)
104
How many types of RNA polymerase do prok and euks have?
```
PROK: Only one type of RNA polymerase
EUK:
3 diff types of RNA pol.
- RNA pol I
- RNA pol II
- RNA pol III
```
105
What is the function of RNA pol I in euk?
- Transcribes rRNA genes (ribosomes)
106
What is the function of RNA pol II in euk?
- Produces mRNA and transcribes some snRNA genes miRNAs
107
How does CDT (carboxyl terminal domain) play a central role with RNA polymerase II?
- Helps coordinate all 3 processing events; 5' capping, 3' poly A tail, splicing
- When making protein, N terminus comes out first and C terminus comes out last
108
In Euk, what is the function of RNA polymerase III?
- Transcribes most small RNA genes (tRNA, some snRNAs, 5S rRNA)
109
What is the ground state of genes in prok and euk?
PROK: Ground state of gene always ON! (always ready to be transcribed)
EUK: Ground state of gene OFF. Silenced genes because DNA in nucleosomes in chromatin in chromosome (so hard to access)
110
How does transcription initiation occur in prok and euk?
PROK: RNA pol. recognising and binding directly to promoter region via sigma factor subunit -35 and -10 regions in E coli promoters
EUK:
Transcription factors binding to promoter (TATA box) and promoter proximal elements
111
What are the specifics of the initiation of trancription in Eukaryotes?
- Recognition involves TATA box (25-30bp upstream from transcription START site)
1. Binding of TATA box binding protein (part of TFIID)
2. Binding causes bending of DNA-landmark to attract other general transcription factors (same for all eukaryotic genes)
112
What is negative control in the context of splicing?
- repressor protein binds to primary RNA transcript and this prevents splicing machinery from removing an intron sequence
113
What is positive control in the context of splicing machinery?
- Splicing machinery is unable to remove a particular intron sequence without assistance from activator protein
114
How are introns recognised and how does eukaryotic cell know where to cut?
- At 5' end, they will have GU and 3' end of every intron will have AG
- They all have branch points ; A base 15-45 bases upstream of the splic site
- Tells you there is same machinery to cut introns out
115
what is the spliceosome?
5snRNAs bound to protein (SnRPS)
(U1, U2, U4, U5, U6)
- cavity is for binding and splicing of mRNA
116
What sites are recognised on introns for splicing?
5'GU and 3'AG
117
What is a SNURP known as ?
The small complex between small nunclear RNA and a protein . "Small Nuclear Ribonuceloprotein"- They are complementary to the GU and A sites
118
What does SNURP U1 have a site complementary to?
- 5' end of intron
119
How many steps are there in the removal of the intron?
- Two splicing steps
120
What is the branchpoint in splicing (intron) known as?
- Conserved A
121
Which U (SNURP) recognises the branch point site A?
- U2
122
What is the difference bwtween INTERCISTRONIC regions and introns?
- INtercistronic separates RNA coding region for protein A from coding region for protein B (BETWEEN genes)
- Intron SPLITS the coding region for one protiein (gene) into two. It is INSIDE the gene.
123
What is the cataylst in splicing?
- snRNA acting as a ribozyme
124
Can intron RNA sometimes catalyse its own excision without proteins or extra RNA molecules?
- YES
125
What is the RNA world hypothesis?
- RNA must have been the genetic material in first cells where it encoded genetic information and catalysed biological reactions
126
What is the function of chaperones?
- Recognise incorrect, off pathway configurations
| - Does this by recognition of hydrophobic surfaces
127
If folding by the chaperone machinery fails, then what is called into play?
- The proteasome; a protein destroying machine (ATP dependent)
