Molecular Flashcards
(297 cards)
1
Q
As well as the linear DNA chromosomes also contain proteins that..(5)
A
- Pack and Unfold the DNA within the nucleus e.g. histones
- Control DNA replication, repair and genetic recombination.
- Maintain chromosome integrity by protecting the end sequences e.g. telomerase
- Govern proper chromosome segregation during cell division
- Regulate gene expression e.g. RNA Primase, RNA polymerase
2
Q
Placement of transcriptionally active genes vs inactive?
A
Activation of a gene is accompanied by migration from the periphery towards the centre of the nucleus
3
Q
In nm the width fibre of chromatin vs when supercoiled?
A
10nm in interphase vs 30nm when supercoiled.- gets so much more densely packed.
4
Q
DNA is wrapped around core.. made of … subunits, to make up a …stabilised by…
A
Histones
8 H subunits
Nucleosome
H1 linker proteins
5
Q
Histone tail names?
A
H2A x2
H2B x2
H3 x2
H4 x2
6
Q
The histone N- terminal tails are rich in ….
Significance?
A
lysines and arginines
Substrate for post-translational control
7
Q
Significance of nucleosome free areas in the DNA?
A
Transcription factors can bind and RNA polymerase so can be translated.
8
Q
What is the centromere? (brief function)
A
DNA sequence in which the kinetochore assembles and mediates chromosome segregation.
9
Q
What is the kinetochore?
A
Protein complex found at the centromere that binds to the microtubules of mitotic spindle.
10
Q
What is the telomere?
A
3 prime overhang repeat of TTAGGG, that maintains integrity and stops the shortening. It’s synthesised by the telomerase.
11
Q
Why is telomere necessary?
A
Without there would be bases lost from the DNA during replication. DNA polymerase needs an RNA primer to elongate from, and at the 3prime end without when this is removed there would be a loss of bases.
Normally can use DNA polymerase to seal the gap, but there is no 5 prime for it to extend from, whereas with telomere another okazaki fragment can be added, and this gap sealed as normal by DNA ligase.
12
Q
Why does the telomere not shorten over time?
A
It is maintained by telomerase. This is an enzyme which is a ribonucleoprotein. This acts as a template, with the sequence AAUCCCAAU, antisense to TTAGGGTTA. The first AAU binds to the 3prime strand, and using this as a DNA template strand adds GGGTTA. It then moves along with AAU binding so adding the next GGGTTA etc, adding 6 nucleotides at a time.
13
Q
What is the centromere structure?
A
Repeated sequence making up alpha satellites, forming condensed transcriptionally inert heterochromatin.
Inner: connect so hold sister chromosomes together (cohesin to)- normal H3 histones, but DNA is demethylated at lysine 4.
Outer: connect to kinetochore- centromere specificH3 histones.
14
Q
What are alpha satellites?
A
In the centromere these make up the DNA repeats that are docking sites for the kinetochore inner plate proteins, and outer plate to that, which the spindle Microtubules attach to.
15
Q
How is the alpha satellite DNA structure different? Why?
A
The heterochromatin contains centromere specific H3 histones (Centromeric protein a variety specifically).
These help hold the sister chromatids together by adhesion molecules ensuring correct pairing.
Outer DNA is demethylated at lysine 4 but normal H3
16
Q
What structure does the yeast kinetochore have?
A
It is basket in shape, which links one single nucleosome in the kinetochore to a microtubule..
17
Q
Only …..% of eukaryotic genome codes for cellular proteins, and …% is just repeated sequence elements.
A
1.5%
50%
18
Q
What are DNA transposons?
A
Mobile genetic elements that jump around the genome using the cut and paste method.
e.g. P-element in fly, Ac-Ds in Maize
19
Q
How does the DNA transposons move in the genome?
A
Cut and paste method:
Transposase enzyme bind to cut the transposon out of the genome.
They are bundled into a mobile transposome.
This can be inserted into another location.
20
Q
Problem with DNA Transposons?
A
They are powerful mutagens as they can change alleles (e.g. Ac-Ds colour change in the Maize when first discovered) or cause damaging mutations.
21
Q
What are retroviral-like transposable elements? Insert into genome how?
A
E.g. HIV.
Viruses that use RNA transcriptase to convert their RNA into DNA which can be inserted into a genome.
22
Q
How do retroviral-like transposable elements Insert into the genome?
A
E.g. HIV
Once the virus is in a host cell, it unenvelopes. The reverse transcriptase of the virus is used to conver the RNA into DNA (adds a DNA strand to the template RNA then this is uses as a template etc and Double stranded DNA is made)
This is then inserted into the host genome. This gene is then transcriped and translated into other viruses (RNA copies, envelope proteins, capsid proteins and reverse transcriptase) so the new virus can leave this host cell and invade another, inserting the DNA into other genome locations.
23
Q
What are non-retroviral Poly-A retrotransposons?
A
The most abundant type, replicate via RNA intermediate using our own reverse transcriptase to replicate via copy and paste into the genome.
24
Q
How to non-retroviral poly-A retrotransposons replicate?
A
The gene e.g. L1 element is transcribed into RNA. Our native endonuclease cleaves the new location DNA. Using the newly created 3prime as a primer and the RNA as a template, the top strand of nicked DNA is extended by our Reverse transcriptase. As this DNA strand is replicated normally, a new double strand with the retrotransposon is created. (page 291)
25
Summary of the difference between the three types of transposible elements?
1. DNA Transposons- use cut and paste by transposase to move (in a mobile transposome) e.g. Ac-Ds
2. Retroviral-like retrotransposons- e.g. HIV- code for all it needs e.g. reverse transcriptase, envelope and capsid and RNA
3. Non-retroviral retrotransposons- e.g. L1 element, use our own enzymes, Nicks the DNA and uses its RNA as a template. Don;t need viral packing.
26
non-retroviral retrotransposons in disease?
Haemophilia
L1 or LINE-1, Long interspersed nuclear element
An insertion of this L1 element into the clotting protein Factor VIII can cause.
27
DNA topoisomersase function?
It prevents DNA Becoming tangled, reducing superhelical tension which is created due to unwinding during DNA replication.
28
How does DNA Topoisomerase work?
Type 1: Nicks and reseals only one strand by unwinding one around the other.
Type 2: Nicks and reseals both strands- more risky and ATP required.
29
DNA replication steps? (6)
1. DNA helicase uses ATP to unwind the DNA and SSB proteins keep the strands apart.
2. RNA primer is added by DNA primase.
3. DNA polymerase adds the new bases from the primer in a 5'-3' direction. This is continuous on the leading strand but for the lagging Okazaki fragments are added.
4. Ribonuclease H removes the primer.
5. DNA polymerase extends over the gap.
6. DNA ligase seals the nick, using ATP.
30
What is the equation for DNA replication/ synthesis?
dNMPn + dNTP= dNMPn+1 + 2pi
dNMPn (Deoxyribonucleotide mono-phosphate Existing strand)
dNTP (Deo... triose phosphate- incoming nucleotide)
31
How is 2pi released from DNA replication/ synthesis?
The incoming nucleotide has three phosphates, so as it joins two are lost.
Initially this molecule is made of two Phosphates and 6 oxygen molecules and termed Pyrophosphate.
Pyrophosphatase then converts this (PPi) to 2 inorganic phosphates and ATP.
32
Where does the ATP needed for DNA ligase come from?
The breaking down of pyrophosphate to 2pi and ATP by pyrophosphatase.
33
Mutations in DNA helicase?
Werner Syndrome- premature aging
| Bloom syndrome- short stature and predisposition to cancer
34
How does the processivity of DNA polymerase increase?
When it is associated with the sliding clamp, which is kept at the primer: template junction by a clamp loader.
ATP is bound to the DNA here also, and when DNA polymerase binds AMP+Pi is given off.
The association of the sliding clamp stops the polymerase falling off the DNA, but allows movement.
It is released at the 5' of the proceeding Okazaki.
35
Use of the SSB proteins?
Single stranded binding proteins, keep the DNA open, and straighten out the chain without covering the bases, stopping hairpin helices forming.
36
How is a replication origin determined?
Destination determined in yeast by where there is an autonomously replicating sequence ARS element.
In humnas, near to certain genes e,g HBB or MYC, but no specific sequence just where there is a nucleosome free zone.
37
After determined where how does a replication origin open? (4 steps)
Formation of a Pre-replicative complex in the G1 phase:
1. Origin recognition complex (ORC) binds to the replication sequence. This lays down the foundations for the Pre-replication complex.
2. Helicase loading proteins bind to the ORC- cdc6 and CdE1.
3. The helicase Mcm 2-7 binds to complete the formation of the Pre-RC.
4. Origin activation- unwinding of DNA and recruitment of DNA polymerase in the S phase.
38
How is it decided whether the Pre-RC formation is activated etc? Why?
Cdk high: Current Pre-RC formation activated, but new Pre-RC formation is prevented
Cdk low: New Pre-RC formed, and any existing not activated.
This prevents more than one origin of replication opening at one time.
39
What is a hydrolysis reaction?
The separation of two molecules by using H2O
40
opposite of a hydrolysis reaction?
Condensation- two molecules join together and H20 is released.
41
What is an oxidation reaction?
Gain of oxygen (or loss of electrons)
42
The four bases can be mutated by which 4 reactions?
hydrolysis, condensation, methylation or oxidation
43
Deamination of which base causes what?
Cytosine is deaminated to uracil. NH3 and H20 are released.
44
Why is deamination of ... base bad?
Deamination of cytosine causes it to become uracil, so when DNA replicates there will be an Adenosine inserted instead of a Guanine.
45
What is a UV pyrimidine Dimer?
Covalent linkage between two carbon atoms in adjacent pyramidines e.g. Thymine dimers or Tymine-cytosine etc,
46
Why is a UV pyramidine Dimer bad?
During DNA replication, there will be either a deletion, subsititution or arrest replication as DNA polymerase cant bind.
47
What is the impact of ultraviolet radiation on DNA?
Can cause Pyramidine dimers to form
48
What is depurination?
Removal of a purine, e.g. A or G, causing a base deletion.
49
What is ionizing radiation?
Double stranded DNA breaks, which are prone to degradation as they have to telemeres, then causing the loss of sequences.
50
What is a PH domain?
Involved in membrane binding and anchoring proteins to the membrane, proteins interract with the charged head of phospholipids
51
How do you experimentally get an epitope tagged protein?
Insert DNA of tag e.g. DYKDDDDK into the protein of interest DNA, introduce this into a cell and it will translate the protein with the tag, which can be purified.
52
Steps for an immunopull down to show interracting proteins?
1. Tag the protein of interest with an epitope.
2. Add other proteins to bind
3. Use specific antibody to the epitope of the POI.
4. Add protein A coated beads complimentary to the antibody (immunoprecipitation)
5. centrifuge to recover the complex.
6. identify the bound proteins by mass spectrometry or western immunoblotting.
53
What is an epitope tagged protein?
A protein tagged with a sequence known to us, so we can purify by using an antibody to the epitope.
54
What are these?
HA peptide YPYDVPDYA
Myc peptide EQKLISEEDL
Flag peptide DYKDDDDK
Common epitope tags for the POI for immunoprecipitation
55
What are these?
Glutathione-S-transferase (GST)
Hexa-histidine (6xHis)
Common protein tags for affinity chromotography.
56
GST affinity pull-down method?
Instead of an epitope being added in immunopull down, GST is added to the POI. The POI then binds to the Glutathione coated beads.
Other proteins are eluted, while the POI and bound proteins are later.
SDS PAGE or western blotting with antibodies are used to separate, or mass spectrometry to identify.
57
What is depurination?
Loss of a purine (A or G- Get pure drugs if u are A G)
| Deletion.
58
Why is depurination bad?
When DNA replication happens a base will then be exised and lost.
59
What is deamination?
Deamination of a pyramidine, thus converting it to anotjer base, e.g. Cytosol deaminated to uracil.
NH3 and H20 given off.
60
What can ultra violet light cause to happen to bases in DNA?
UV pyramidine dimers.
| Covalent linkage between 2 carbon atoms in adjacent pyramidines e.g. thymine dimers or tymine cytosol etc.
61
WHat is bad about UV pyramidine dimers?
Leads to either a base pair deletion, substitution or can arrest the DNA replication as DNA polymerase cant bind.
62
What can ionizing radiation do to DNA?
Cause double stranded breaks which then have exposed ends that are prone to degradation. They have to telemeres so can result in the loss of sequences before repaired.
63
Two methods for repairing ionizing radiation breaks in DNA?
Non-homologous end joining and Homologous recomibation.
64
WHat is the difference between the two methods of reparing ionizing radiation?
Non-homologous end joining is musch faster but less precise can result in loss of bases, whereas homologous recomination is the last line of defence and is more accurite.
65
How does non-homologous end joining work?
Quickly joins up the two broken ends of DNA, but this can cause loss of DNA due to degradation.
66
How does homologous recomination work after a DNA break? (short description)
Sister chromatids are recruited (often near just after replication anyway) and used to ensure there is no DNA loss. These are used as templates to repair any lost DNA bases.
67
How are sister chromatids used to repair DNA in homologous recomination?
1. Exonuclease degrades the 5prime ends of the cut DNA back, to create resected sticky ends.
2. DNA binding protein RecA promotes strand invasion (opening the sister chromatids strands so that he complimentary broken strand can bind)
3. A heteroduplex is created (a DS molecule created by recomination from a different source- in this case two sister chromatids).
4. A holiday junction is also created (crossing of the DNA)
5. DNA polymerase synthesises new DNA using the sister chromatids as templates.
6. The invading (damaged) strand is released nd the broken double helix of both reforms with DNA Helicase
7. DNA synthesis continues using the repaired strand as a template.
8. DNA ligase seals the nicks created.
68
What is base excision repair after?
After deamination of cytosol to uracil
69
Method of base excision repair?
After deamination c to U.
1. The incorrect uracil base is recognised by DNA glycosylase and removed.
2. AP endonuclease removes the sugar and Phosphodiesterase removes the phosphate so there is a gap in the DNA
3. DNA polymerase adds the new nucleotide using the other strand as a template.
4. DNA ligase seals the nick.
70
What is nucleotide Excision Repair used after?
Pyramidine dimer formation.
71
What is nucleotide excision repair method?
1. Dimer recognised by Excision nuclease and the backbone of the DNA is cleaved a few bases either side of the dimer damage.
2. DNA helicase removes the sliced single stranded fragment.
3. DNA polymerase extends from the Primer:template junction using the other strand as a template.
4. DNA ligase seals the nick.
72
What method is used to repair deamination of a DNA base?
Base Excision repair. (B and D both early in alphabet, whereas N and P later)
73
What method is used to repair a pyramidine dimer in DNA?
Nucleotide excsion repair. (B and D both early in alphabet, whereas N and P later)
74
Difference between endo and exonuclease?
Exonuclease removes nucleotides as the end of a DNA strand, whereas Endonuclease is in the middle.
75
What is a holliday junction?
A crossed structure created during recombination, where the DNA strands are separated so one can cross and bind to complimentary bases of another.
76
Enzyme that promotes strand invasion?
RecA
77
Method of homologous recomination? (non repairing)
1. Spo11 Endonuclease makes the initial cleavage in the middle of the strands.
2. Mre11 Exonuclease resects the 5 prime end degrading it to create a staggered double stranded break.
3. Strand invasion catalysed by RecA.
4. DNA synthesis catalysed by the DNA polymerase, and second strand captured by the strand invading, to make a double holliday junction.
5. (look at picture) cuts and ligations of strands happens so recombination occurs.
78
What are the phases of the cell cycle?
Go- Gap phase where cell cycle is arrested
G1- Gap phase 1, checking environment is stable before going into the cell cycle.
S- Synthesis, interphase, all DNA is replicated.
G2- Gap phase two checking all of the DNA is replicated and envronemnt still favourable.
M- Mitosis, Segregation of the chromosomes into separate cells and cytokinesis.
79
Phases of Mitosis?
Interphase (DNA replication)
Prophase (Condensation of sister chromatids)
Prometaphase
Metaphase (mitotic spindle attatch to the kinetochore)
Anaphase (separation of chromatids)
Telephase (envelope reforms)
80
Two types of yeast replication?
Budding yeast: A bud forms off the yeast, and duplicate contents and this stretches into the bud which separates off into a new cell.
Fission yeast: Normal eukaryote way
81
Advantages of using yeast to study cell cycle?
- Rapid divisions once every hour
- Can be done haploid or diploid
- Cell cycle genes are highly conserved so good for studying our own mechanism
82
Why is an Xenopus a good biochemical model for cell cycle studying?
- easy to collect eggs
- Rapid division rate (every 30mins)
- Large size good for collecting purified proteins
- Can do RNA injection experiments on the oocyctes.
83
How would one experimentally do cell-free mitosis? Why?
Extract the cytoplasm of an egg cell, add the sperm to fertilise in a test tube.
Add ATP.
Can then add antibodies which can then target proteins in the solution and analyse the proteins present at each stage.
84
What are the three checkpoints in mitosis?
G1 to S- Is the environment favourable?
S to Prophase- Is all the DNA replicated? (negative signals)
Metaphase to Anaphase APC: Are all the chromosomes attatched to the spindle?
85
What varies to promote the cell cycle?
Cyclins. Cyclin levels vary and these bind to cdk's (cyclin dependent kinases).
CDK's then phosphorylate many proteins that are specific to certain stages of the cell cycle.
86
How are CDK's regulated apart from Cyclins binding?
On phosphate activates, two inactivates.
Wee1 was phosphorylate in the inhibitory position to inactivate.
CDK 25 phosphatase can remove this nand activate it again.
p27 can also inactivate.
87
What is Anaphase promoting complex?
It is a ubiquitin ligase which ubiquitinises M and S cyclin, so promoting Anaphase.
The ubiquitination is then achieved by E1 and E2 enzymes under APC regulation.
Cdc20 activates APC.
88
APC also ubiquitinates ..... as well as the cyclins. why?
securin for destruction, enabling the eventual destruction of cohesin and thus sister chromatid separation
89
Difference between meisos and mitosis?
Homologue chromosomes, materal and paternal segregate, to give only half the total number= haploid 23 instead of 46.
90
How is gentic diversity increased further? (beyond just random assortment of chromosomes?)
Crossing over. Bivalents are formed which allow genetic recomination.
91
When in meiosis do the different chromatids separate?
Anaphase 1: The sister chromatids segregate.
| Anaphase 2: Paternal and Maternal chromosomes segregate.
92
What facilitates the pairing of a bivalent?
Synaptonemal complex made of cohesin between the maternal and paternal chromosomes.
and base pairing of homologues.
93
Crossing over of chromosomes happens at a .....?
chiasma
94
Two reasons for homologous recombination?
1. Align the chromosomes up ready for anaphase and facilitates the formation of the synaptonemal
2. Allows for genetic recombination between paternal and maternal DNA on the same chromosome.
95
Word for abnormal number of chromosomes?
Aneuploid
96
...% of sperm are aneuploid and ...% of eggs
4% sperm
20% egg
causes miscarriage/ downsyndrome (philadelphia chromosome)
97
Where would the kinetochore have to attatch to the spindle to secregate differently in meiosis?
```
In mitosis (and Meiosis 2) connected to each sister chromatid so the spindles pull them apart.
In meiosis 1 they are attatched to the centre of two sister chromatids so they segregate together, and maternal and paternal segregate.
```
98
DNA dye that shows nuclei?
DAPI binds to A-T rich DNA causing it to fluoresce
99
How can T be seen in DNA replication?
BrDU is a thymidine analogue that subs for the base and can be detected by fluorescent antibodies.
100
Pulse treatment can be used to tell which cells are what?
In the S-phase of the cell cycle.
101
How do cyclins activate CDK?
Cause a conformational change
102
How are different proteins expressed when all cells have the same genome?
Transcriptional regulation, not all genes are transcribed, and therefore translated into proteins. Even after this the proteins may be modified etc to be different.
103
DNA binding proteins charge? Reach into the ..... of DNA
Positively charged Binding AA's (DNA negatively charged due to the phosphate backbone e.g. gel electrophoresis
DNA major groove
104
DNA binding proteins will interract with the ...... of the DNA in the major groove (4 details)
response element in the major groove- makesspecific interractions with the bases via hydrogen bonds and non-specific to the backbone due to the charge
105
On a molecular level how do DNA binding proteins e.g. transcription factors bind to the DNA response element?
Side chains of the bases are either hydrogen donors, acceptors, a hydrogen molecule or a methyl group, which can interract with amino acids of the transcriptionf actors
e.g. asparagine binding to adenine via two hydrogen bonds.
106
What are examples of DNA binding protein types?
Transcription factors, polymerases, nucleases,
| e.g. via zinc fingers, leucine zippers, helix-loop- helix etc
107
Example of yeast transcription factor? Binds?
Rox1, known to bind to 8 sites in 3 yeast genes, which all have different affinity to the protein. (3 in HEM13, 4 in ANB1 and Rox1 tiself)
108
How can different affinities of response elepments to a transcription factor cause different levels of transcription?
E.g. ROX1 doesnt bind perfectly, so is on some of the time and off other times, and the amount of transcription is the ratio of time on vs time off, depending of its affinity to the response element.
109
What is a consensus sequence?
The average sequence that a DNA binding protein will bind to e.g. the most common bases at those positions
sequence logo uses this to create an image of the text with the more common bases appearing larger than less common ones.
110
What are permissive transcription factors also called?
General transcription factors
111
How do transcription factors regulate DNA transcription? (3)
1. Interracting with the RNA polymerase complex to promote or repress
2. Altering acetlyation of the DNA, to make it more or less accessible for transcription.
3. Binding to other transcription factors
112
How does DNA looping occur?
Chromatin doesnt bend easily so for two proteins to interract and loop they have to be more than 500bp's away.
113
How is the promiscuous nature of the enhancers modulated?
(that they can work at enhancing more than one gene) By insulator elements, these block this and prevent enhancers activating downstream genes.
114
Two types of insulator elements?
an enhancer-blocking function and a heterochromatin barrier function.
115
There may be many inputs of transcription regulation, how is this interpreted?
It is interpreted as a sum of all of the transcription activators and repressors, and whether these are strong or weak, to give the final level of transcription.
116
How does tryptophan regulate its own transcription?
If high the tryptophan repressor protein undergoes a conformational change so can bind to the DNA and represses proteins required for tryptophan synthesis, whereas if low tryptophan in the cytoplasm this protein is not bound to genes actve.
117
7 ways that transcription factors can be activated?
1. Ligand binding
2. Phosphorylation
3. Release from the membran, separate from the TM part.
4. unmasking, removal of an inhibting subunit
5. Removal of a signal peptide so can go into the nucleus
6. Addition of a second subunit
7. Protein synthesis
118
Transcription factors can act ...... to activate transcription the most by?
synergistically
Binding together helps prevent them falling off,
or the first may say unwind the DNA so the second can bind.
119
Epigentic changes are periminant/non-perminant?
Non-perminant
| they are not in the germ line, but can persist through life.
120
On where do the epigenetic modifications occur?
On the Lysine rich N terminal tails of the core histones.
121
Acetylation impact on transcription? By which enzyme?
Upregulates transcription by Histone acetyltransferase.
122
How does acetylation exert its effect on transcription?
Acetylation of the lysines creates sites for transcriptional activators containing a Bromodomain to bind.
123
Which epigenetic modifications are mono, which di and tri?
Acetylation is mono (single modification),
| whereas methylation can be mono, di or tri, all to one tail.
124
Acetylation and methylation can/can't occur together?
Can't- they are mutually exclusive
125
What enzyme causes acetylation, and which reverses it?
```
Histone acetyltransferase (HAT)
reversed by Histone deacetylase
```
126
What enzyme causes methylation and which reverses it?
Histone methyltransferase
| reversed by Histone demethylase
127
Does methylation activate or repress transcription?
```
If on:
H3-K4- activates
H3-R17- activates
H3-K9- inactivates
H3-K27- inactivates
(where K is lysine, and R is arginine)
Think: Meth can be bad
```
128
How does methylation cause the effect on transcription?
Depending on where the methylation is, repressors with chromodomains or activators with PHD zinc finger domains bind.
129
Writers or readers of epigenetic modifications?
Writers are the enzymes e.g. Histone Acetyltransferase, or Methyltransferase,
the readers are the binding proteins e.g. activator or repressors.
130
What evidence is there that acetylation is associated with transcriptionally active sequences?
Metagene analysis of distribution of histones across genes in the genome- shows a link with transcription
131
What remodels chromatin to make it more accessible?
nucleosome remodelling complexes.
| heterochromatin to euchromatin
132
Which is the more tightly bound chromatin?
Heterochromatin (if heterosexual harder to reproduce as still single, whereas euchromatin with u only so can form a family and translate proteins)
133
How do transcriptional activator proteins that work on chromatin? (4 ways)
Nucleosome remodelling complexes turn into more accessable euchromatin then..
Histone chaperones can remove histones, or replace the histones with different modified histones.
Or histone modifying enzyme recruits code readers and writers (epigenetic)
Often combination of these which recruit RNA polymerase.
134
What enzyme removes histones from the nucleosome?
Histone chaperones
135
How can transcriptional repressors work? (6)
- Prevent activators from binding
- Mask the activation site
- Directly interact with the general transcription factors to turn them off
- Recruit chromatin remodelling factors that package the DNA into heterochromatin.
- Removal of acetylation- Histone deacetylase.
- Remove methylation or add depending on location (histone demethylation, or methyltransferase)
136
What is the example of methylation in Drosophila?
The polycomb proteins can repress transcription.
The PRC2 complex is recruited and a component of this (Enhancer of zeste Ezhz) is a histone methylationtransferase enzyme, causes the methylation of H3 at k27 (lysine)
137
Effect of the methylation in the Drosophila example?
The H3K27 Methylation is recognised by the code reader PRC1 which has a polycomb chromodomain, causes the remodelling of the chromatin into heterochromatin, so transcription is repressed.
138
How does the calico cat show epigenesis?
Only females can be calico, as is on the X chromosome so males will end up with only one of either the black coat colour or orange.
Where females can have both expressed, random epigenesis in early embyrogenesis occurs to only express either or, thus creating the spotty patched appearance. (random x linked inactivation of whole chromosome in each somatic cell)
139
How does X inactivation happen?
Synthesis of a non-coding RNA (xist) from the X-inactivation centre (XIC) on the chromosome desined for inactivation.
Xist binds to the chromosome for inacitvation stimulating its own production so accumulates around.
Xist accumulation causes condensation of the chromosome into a transcriptionally inert chromosomal structure.
140
How does Xist cause the condensation of the chromosome in x-inactivation?
It recruits histone modifying enzymes that cause chromosome condensing and other polycomb groups leading to H3K27 and H3K9 methylation.
141
What is the inactivated X chromosome structure like after?
Barr-body visible in all somatic female chromosomes.
142
How is RNA different from DNA?
```
Ribose not deoxyribose (H instead of OH)
Uracil not Thymine base
SIngle strand usually- if mRNA
RNA very unstable.
Non Watson and Crick pairing, with a wobble base in the third position.
TRNA in clover shape with anticodon
```
143
Three types of RNA polymerase?
RNA polymerase I- Ribosomal RNA
RNA polymerase II- Our cells use for RNA transcription
RNA polymerase III: tRNA, SiRNA, housekeeping and rRNA
144
Transcription does/doesnt need an RNA primer?
Doesnt
145
Problem encountered with transcription? Solved by?
DNA cannot spin, so gets superhelical tension as it rotates every 10bps. This conformation is energetically more favourable but is harder to open.
Topoisomerase releases this.
146
How does RNA polymerase know where to start?
TATA region is normally around 30pbs up from the start point, and BRE around 35.
147
What needs to bind to initite transcription of mRNA?
General transcription factors bind at the TATA region and distort the DNA bringing proteins closer so can assemble.
Protein complex is needed for transcription to start, including RNA polymerase, chromatin remodelling complex, histone modifying enzyme etc.
148
WHat 3 things have to be done in order to create a mature mRNA?
1. Introns spliced out
2. Capping 5prime- increases stability, and help ribosomes to bind
3. Polyadenylation of 3prime- Part of termination process, helps mRNA for nuclear export and translation.
149
Splicing of mRNA is catalysed by what enzyme? Structure?
A spliceosome
Made of 5x snRNAs U1,U2,U4,U5,U6.
These bind with other proteins to make the snRNP's which interract with the mRNA, and form the core of the spliceosome.
150
unspliced mRNA is called what?
pre-mRNA
151
Splicing out introns of mRNA is specific to?
eukaryote mRNA
152
How is an intron spliced out mRNA?
1. SnRNP's combine with the pre-mRNA and binds snRNA's to form a spliceosome.
2. GU- donor site at the 5prime end of the intron is cut.
3. AG- acceptor site on the 3prime of the intron is cut.
4. There is an A in the middle of the intron= the branch point. This starts attacking the phoshodiester bond on the 3 prime of the donor site.
5. The 5 prime donor site end joins with the branch point and a lariat is formed.
6. The 3' OH of the donor sie attacks the phosphodiester bond on the acceptor, forming a free lariat.
7. Exon sequence is joined back together
8. Lariat is degraded in the nucleus.
153
Function of the spliceosome?
1. Recognise the 5prime donor and branch sites.
2. Bring these sites together
3. Catalyse RNA cleavage.
154
5prime capping happens in...?
All eukaryote mRNA. When 20-40 nucleotides long.
155
What is unusual about 5 prime capping?
It is a 5' linkage to another 5' of Guanosine (Guanine attratched to Ribose)
This guanine is then methylated.
156
What molecule is at the 3' end of DNA/RNA? 5'?
5' ends in P Phosphate
| 3' ends in OH hydroxyl
157
How does 5' capping happen?
1. RNA triphosphatase cleaves the 5' mRNA to leave '5' diphosphate' instead of triphosphate.
2. Guanylyltransferase adds a Guanine monophosphate group, giving off a Phosphate. (still leaving 3 phosphates in a row)
3. Methyltransferase methylates the guanine.
158
Method of Polyadenylation?
1. As the the mRNA is being translated by RNA polymerase II, the pol II c-terminal domain (CTD) is highly phosphorylated and mimics RNA attracting the factors neededed to the polyA tail.
2. CstF and CPSF recognise and bind to the polyA signal AAUAAA of the mRNA as it is being transcibed by RNA polymerase II. (cleavage stimulating factor and
Cleavage and Polyadenylation Specific factor) These recruit other proteins needed.
3. PolyA polymerase is added to the 3' end and add 'adenines to the 3'. PolyA binding proteins aid.
159
What is polyadenylation?
The addition of upto 200 Adenine bases by polyA polymerase
160
Where is the PolyA signal in mRNA?
This AAUAAA signal is only 10-30 nucleotides downstream of this signal is the cleavage site (STOP codon), so is near the 3prime.
161
4 alternative splicing options?
(optional) Exon skipping: One exon may be cut out with the introns as they are spliced
(optional) Intron retention: One intron may be kept
Mutually exclusive Exons: Either one exon or another will be spliced, never both present or absent.
Internal Splice site: Only half of the intron is removed.
162
How are different isoforms of mRNA be formed?
Alternative splicing differences, can remove certain exons in the process of removing introns.
163
What two types of internal splice sites are there?
```
Alternative donor (5') site
Alternative acceptor (3') site
depending on which side of the intron half is spliced.
```
164
Example of alternative splicing in Drosophila sex determination? Male:
In the male: Sx1 (sex-lethal) and tra (transformer) transcripts are spliced so give a nonfunctioning protein.
Dsx (double sex) however is spliced to remove an exon (as the internal intron splice sites are inacitvated) and this gives rise to a DSX protein which represses genes required for female development.
Therefore MALE.
165
Example of alternative splicing in Drosophila sex determination? Female:
Two chromosomes in the female causes a small amount of functional SX1 to be made, which is an RNA binding protein that binds to its own transcripts and blocks this inactivating splice site from the spliceosome (U2AF) so more SX1 is made.
SX1 also acts on Transformer to block the splicing here also, giving a functional transformer protein.
Tra interracts with Tra2 and they bind and activate a different splice site in DSX, giving a different C terminus. This leads to a different isoform being created, which represses male specific genes.
166
C terminus? N terminus?
Carboxyl- COOH at the end.
| NH3- at beginning
167
Example of Polyadenylation on mRNA changing the function of a protein?
B lymphocyte can produce 2 antibody isoforms, either a membrane bound form or a secreted antibody (mature).
Long transcript: The first stop codon located in an intron is spliced out so the full length is translated, including the transmembrane domain portion.
Short transcript: The intron is not spliced, so the translation terminates at the first STOP codon, without the transmembrane portion, so the antibody can be secreted.
168
What is leaky scanning?
Can have 1 or even 2 AUG start codons that are ignored by the ribosome, by having an optimal sequence of AUG. e.g. in a Kozak sequence.
These will all have the same reading frame so its just the N terminus that will differ.
169
Why is translation more commonly started at the first AUG?
High levels of EIF-4F favour the first AUG.
170
WHy do retoviruses such as HIV need to be spliced?
In order to be secreted out of the nucleus the HIV mRNA needs to be spliced (or would be retained and degraded), but this would cause a loss of information.
171
How do retroviruses such as HIV overcome splicing?
The mRNA is spliced so it can leave the nucleus, and this form is translated into Rev and other viral proteins, which go back into the nucleus and bind to the Rev responsive element of the full length mRNA. The Rev protein interracts with nuclear export receptor Crm1 and allows movemnt of the full mRNA, inspite of the present intron sequences.
172
How can HIV levels be detected?
Can monitor HIV infection by detecting Rev levels. These allow trafficking of the full length mRNA out of the nucleus to be translated into more viruses.
173
What are UTR's?
Untranslated regions of an mRNA. They can form into stem loops which are recognised by certain cellular proteins or can act as a signal sequence.
174
Two functional examples of UTRs untranslated regions in mRNA?
They can form into stem loops which are recognised by certain cellular proteins.
1. e.g. Acconitase to ferritin and transferrin stem loops, in iron regulation
e. g. Bicoid attatched to the cytoskeleton at the anterior tip of embryo.
175
Function of Ferritin and transferrin?
Ferrin: Stores iron, therefore reducing the amount available.
Transferrin: Channel that brings iron into the cell, so increases iron levels.
176
When there is low iron levels in the cell?
Acconitase binds to the 5' Ferritin and the 3' transferrin stem loops .
This blocks translation initiation of Ferritin, but stabilising the degredation of Tranferrin by blocking the endonuclease cleavage site.
Therefore Ferittin levels decrease (less storage) and Transferrin increases (more ion brought in) SO IRON INCREASES.
177
When high iron levels in the cell?
Iron binds to acconitase, which causes a conformational change so disociates from tranferrin and ferritin.
Translation initiation complex recruited to Ferritin 3' UTR- more is made (so more iron stored)
Transferrin degraded by endonucleolytic cleavage (less brought into the cell)
IRON LEVELS DECREASE
178
When and by whom was the structure of DNA found?
1953- Watson and Crick
179
Who found evidence that DNA carries genetic info?
1944-Avery
180
Start and stop codons?
Start; AUG
| Stop: UAA UAG, UGA (U Are An Ugly And Grotesque University Gorilla Amber)
181
How is the Amino Acid bound to the tRNA? Enzyme?
The synthetase primes the amino acid first by adding AMP to the C terminus. Ribose-O-AA
It then uses the adenylated AA to form Aminoacyl-tRNA.
Ester bond by Aminoacyl tRNA- synthetase
This hydrolyses ATP, and two inorganic phosphates are given off.
182
What can be different about the bases in tRNA?
Uracil may be modified to Pseudouridine, or 2H added to make Dihydrouridine.
Adenosine can be modified in the wobble position to Inosine which can pair with U,C, or A
183
How is Protein synthesis initiated?
1. Small ribosomal subunit searches for AUG where tRNA is bound.
2. Met tRNA binds with eIf-2 to the small ribosomal subunit.
3. If Incorrect binding: No large subunit binds, and EIF2 doesnt dissociate as associated ATP isnt converted to ADP. After time, the tRNA falls off.
4. If correct binding: The EIF-2 dissociates as the ribosome is a ribozyme which catalyses the convertion of ATP to ADP, so the large ribosomal subunit can associate.
184
How does translation of proteins happen?
1. Incoming tRNA is associated with elongation factor EF-Tu-GTP.
2. Two lags; EF-Tu GTP needs to be converted to GDP and this protein needs to dissociate, allowing time for incorrect tRNA's to fall off.
3. If correct binding GTPase activity is faster and the AA tRNA is captured. Whereas if incorrect no GTPase activity.
4. This hydrolysis of GTP, provides the headgrowth energy for the peptide bond.
5. EF-G in GTP form binds close to the A site and the hydrolysis causes a tRNA shape change which promotes movement of the tRNA to the next site (A to P or P to E).
6. Using the EF-TU head growth energy a peptide bond is created by peptidyl transferase between the C terminus of the growing polypeptide and the AA on the tRNA.
185
How does termination of translation occur?
1. Stop codon UAA UAG UGA encountered and the tRNA doesn't recognise.
2. Releasing factors bind to the A site of the ribosome.
3. Peptidyl tranferase catalyses the transfer of water to the C terminus of the polypeptide.
4. Addition of water results in the dissociation of the polypeptide from the ribosome
5. Presence of the releasing factor promotes ribosome disassembly.
186
Structure of tRNA?
```
3' Amino acid joins to.
D loop
Anticodon loop
T looop
5'
```
187
How is the tRNA charged?
AMP is added to the C terminus by aminoacyl synthetase. creating adenylated Amino acid.
The phosphate carries the high energy charge.
called an Aminoacyl-tRNA, and ATP hydrolysis energy is contained in the ester bond.
188
How is it ensured that the correct AA is joined to tRNA?
Two adapters ensure.
The specific synthetase- the AA and tRNA both need to fit in the specific pockets within the enzyme. Has a higher affinity to these. Then the matching of the anticodon and AA.
The tRNA that matches to the correct codon of the DNA to the correct AA in the ribosome.
189
What can break the high energy bond between AA and tRNA
nitrogen to a low energy state
190
What makes incorrectly paired tRNA's fall off?
The lag time is made longer as the EF-GTP isnt hydrolysed to GDP, giving it tiime to fall off.
191
Subunits of the ribosome function?
Small: Facilitates the tRNA mRNA interaction making sure they match
Large: catalyses the polymerisationn
192
Before translation what is the structure of the mRNA?
In a loop.https://www.brainscape.com/decks/6824026/cards/quick_new_card
EIF-4E is bound to the 5'cap.
This binds to eIF-4G.
This binds to the 3' polyA tail.
193
Why is mRNA held in a loop?
Stabilises it and it acts as a checkpoint for broken DNA.
194
How are proteins folded after translation?
Folded into a molton globule, which hides the hydrophobic side chains, to achieve a lower energy state.
The AA sequence is thought to have evolved to help this formation.
195
How do molecular chaperones work?
e.g. HSP50/60 in high heat.
e.g. HSP60:
Incorrectly folded proteins get stuck in here with the hydrophobic side chains near the entrance and unfold.
GroES cap puts a lid on this using ATP, to seal the protein in the protein for around 15 seconds to give it a chance to refold.
ADP and Pi given off and the correctly folded protein.
196
What is the problem with incorrectly folded proteins?
They can aggregate e.g. Prion proteins- Cross Beta filament amyloid plaques in alzheimers, or Huntingtons
Protease resistant so cant be degraded and even cause other proteins to fold incorrectly.
Aggregate and disrupt cella function.
197
How can translation be regulated?
The conversion of eIF-2 GTP to GDP will premote translation, but if not hydrolysed then the cell can go into quinscence
198
How is eIF-2 recycled? Control?
The GDP needs to dissociate so that GTP can bind and it be reused. EIF-2B is required for this.
When EIF-2 is phosphorylated this is inhibted as it binds to EIF-2B inacitvating it and more, so slowing protein synthesis.
199
How are protein synthesis levels controlled and why?
By phosphorylating EIF-2 by a protein kinase so it binds and inacitavtes EIF-2B so the GDP cannot be dissociated.
If a cell was going into Go stage or infected by a virus.
200
How can protein synthesis be initiated independent of the 5' cap?
Internal Ribosomal Entry sites:
Stem loops in RNA can initiate. The stem loop binds to EIF-4G directly, without the need of EIF-4E. This could result in different exons being transcribed due to another open reading frame being read.
201
When are Internal Ribosomal Entry Sites often used? How regulated?
BY viruses to get their own mRNA translated by blocking the 5' cap translation of the host. They can produce a protease that cleaves hosts EIF-4G so cannot bind to the EIF-4E, but can still bind to the IRES.
202
Why do mRNA's only have a certain life span (half-life)?
Because gradually the polyA tail is degraded from the 3' by exonucleases and when it gets to only 30bps from around 200, it is decapped and destroyed from the 5' end.
203
How is the life span of some mRNAs extended?
They can be readenylated in the cytoplasm, and often factors that promote translation block degradation e.g. EIF-4E competes with DAN which is an exonuclease.
204
Summary of how translation is controlled at the mRNA level? (6 and examples)
1. Alternative splicing e.g. Sex Drosophila, B lymphocyte antibody isoforms
2. Regulated nuclear transport e.g. HIV retoviruses, Rev interracts with Crm1.
3. Untranslated regions UTR's: signal peptides, or regulate translation e.g. Iron homeostasis (acconitase to ferritin, transferrin)
4. EIF-2 Phosphorylation inacitvates EIF-2B needed for recycling by removing GDP.
5. Internal ribsomal entry sites: Stemloops bind EIF-4G to polyA- alernative ORF, e.g. viruses cleave EIF-4G cant bind to EIF-4E
6. Polyadenylation exonuclease degradation gives mRNA half life, limits translation. EIF-4E competes with DAN for binding, promotes life
205
Word meaning both hydrophobic and hydrophillic?
Amphipathic
206
On the cell membrane examples of the glycerol parts? lipids? Structure?
Choline-Glycerol-p-lipid (FA)
Glycerol e.g. Phosphotidyl
Lipids: Ethomolamine, Serine, choline, myelin
or Sphingolipids (no glycerol) usually sphingomyelin
207
Which phospholipid has a net negative charge?
Phosphotidyl-serine, both parts are negative.
208
How is flexibility within the cell membrane acheived?
fully saturated bonds in fatty acids make the membrane very inflexible as is so densely packed, whereas cis double bonds provides kinks making the membrane more flexible
e.g. vegetable oil- omega 3- 3 unsaturated bonds vs lard
209
Examples of unsaturated fatty acid bonds?
Sea plants and fish, nuts= Omega 3- 3 unsaturated F.A's with the first at position 3.
Land plants and animals= Omega 6- 2 cis bonds with first at position 6
210
Function of cholesterol in membrane?
Helps to seal the membrane, 17% of PM content, local decrease in permeability/ fluidity
211
Percentages of composition of the plasma membrane?
24% Phosphotidyl choline
19% spingomylein
17% cholesterol
212
Charge of the plasma membrane?
The inside is negatively charged, so repels other negatively charged intracellular compnents such as vesicles.
213
Where would phosphotidyl serine be found?
Phosophotidylserine is negatively charged and strictly found on the cytosilic side of the plasma membrane.
214
Why is the location of phosphotidyl serine important? how maintained?
```
Phosphotidylserine transferase flippase keeps it on the cytosilic side where scramblase randomly moves them between leaflets.
Because phosphotidylserine (-) signals for apoptosis if on the extracellular side.
```
215
How can phosphotidyl flipover be detected experimentally?
With fluorescent Annexin V test
216
Phosphotidyl composition in the cell membrane?
4% total but 8% total in the cytosillic side.
217
How is cholesterol endocytosed? (9)
1. LDL particles pack cholesterol in the blood
2. LDL particles bind to receptors on the cell membrane
3. LDL particles are endocytosed into a clathrin pit, and make a coated vesicle.
4. Vesicle is uncoated.
5. Fusion of the LDL vesicle with an early endosome.
6. LDL binding to the receptor is broken by a proton pump which acidifies the endosome.
7. LDL receptor is bud off in a transport vesicle back to the cell membrane.
8. LDL buds off in another vesicle and fuses with a lysosome.
9. Lysosome uses hydrolytic enzymes to break down the LDL and free cholesterol is released into the cytoplasm.
218
From LDL binding to the receptor how does the final coated vesicle form? (6)
1. LDL binds to vesicle.
2. Phosphotidylinositol on the inner leaflet of PM is phosphorylated to PIP2 by PI kinase.
3. Adapter proteins bind to the receptor and PIP2.
5. AP2 adapter protein causes a conformational change which exposes more receptors for cargo.
4. Clathrin then binds to the adapter proteins.
5. Gradually more receptors will be recruited and the membrane will invaginate as the tri-legged curvature of clathrin forces this.
6. Dynamin pinches the vesicle off by hydrolysing GTP to GDP.
219
Famililal cause of Atherosclerosis?
LDL Receptor mutation, means it cannot be internalised into cells, so the LDL accumulates in the blood vessels forming plaques.
220
Structure of Clathrin?
Tri-legged, Tri-skeleton made of 3 heavy chains and 3 light. it can link to the actin cytoskeleton to cause vesicular movement.
221
Use of Dyamin? Mutation in?
Pinches off a clathrin coated vesicle by hydrolysing GTP to GDP.
Mutation: causes formation of vesicles on the cell membrane which do not pinch off.
222
Golgi and ER versions of Clathrin?
Through the Golgi: COP1
| From ER: COP II
223
Formation of COPII coated vesicles? (4)
Sar1 is a coat recruitment GTPase
1. Sar1-GDP binds to a Sar1- GEF on the ER membrane causing Sar1 to release its GDP and GTP bind.
2. This GTP binding, triggers a conformational change in Sar1 which causes a helix to insert into the cytoplasmic ER membrane.
3. This causes the membrane bending
4. GTP bound Sar1 binds to COPII adapter proteins and coat proteins.
224
How does phagocytosis happen?(4)
1. A pseudopod of the phagocyte extends around a microbe (by actin treadmilling etc) to engulf it into a phagocytic vesicle.
2. The phagosome is fused with a lysosome to make a phagolysosome.
3. Microbe is degraded and digested by hydrolytic enzymes (acid hydrolase)
4. The residual body is exocytosed out
225
What is autophagy?
Smalller vesicles fusing around a damaged organelle. Then fusion to a lysosome for acid hydrolase to breakdown and recycle the components into the cytosol.
226
3. types of vesicularisation before fusion to a lysosome?
Phagocytosis
Autophagy
Endocytosis
227
SNARE proteins not only sure correct location fusion but also..?
Help to overcome the repulsion of negatively charged components that need to fuse.
228
How do SNARES work?
4 SNARE proteins coil around each other to force two membranes together.
-VSNARE Synaptobrevin (transmembrane)
-T SNARE Syntaxin (transmembrane)
other T SNARE SNAP25 (x2)
229
Strucutre of the 3 SNARES?
VSNARE Synaptobrevin (transmembrane)
-T SNARE Syntaxin (transmembrane)
other T SNARE SNAP25
The transmembrane are hydrophobic AA's forming a helix.
3 form a tight coiled coil on interaction
230
How do SNARES dissociate after joining?
NSF catalyses the dissociation by hydrolysising ATP
231
How does Botulism/ tetanus work?
neurotoxin attacks SNARE proteins so blocks exocytosis of ACh across the cleft at a NMJ.
1. Botulinum endocytosed into pre cleft after binding to gangliosides on the neural membrane.
2. The lightchain of Botulinum escapes (SNARE protease) the vesicle
3. The SNARE protease cleaves SNARES, and so cannot exocytose any vesicles for several months, until resynthesis.
232
Botox how work?
Cleaves SNAP25. Picogram amounts. Causes local muscular paralysis
233
How are proteins helped to be protected form harsh environments and tagging mechanism?
Carbohydrate additions e.g. complex sugars e.g. glycoprotein, glycolipid or proteoglycan (Protein+GAGs)
234
What is a proteoglycan?
A protein bound to a mucopolysaccharide thats heavily glycosylated e.g. Protein+ GAGs
235
Proteins destined for where are made in the Rough ER?
Membrane proteins and secretory proteins
236
Proteins destined for where are made in the Smooth ER?
Nuclear
237
Sequence of passage of a membrane protein for example in the cell?
Rough ER-> smooth ER-> Golgi-> secretory vesicles-> plasma membrane
238
Protein synthesis starts where? Why?
In the cytosol
As there is a pool of ribosomal subunits in the cytosol. And the signal sequence of the peptide that is being made then signals the location of where the protein needs to be synthesised
239
free ribsome cycle?
No signal sequence on the protein so are made in the cytosol and just released into the cytosol.
240
Membrane bound ribsome cycle?
Signal sequence shows the location of where the peptide is destined to go. This is recognised by a signal recognition particle as the protein is being synthesised. This then binds to a signal recognition particle receptor and is attatched to the membrane of destination e.g. ER with the ribosome.
Protein translocator is recruited and the signal can bind while the protein passes through.
The signal is then cleaved off
241
Whats a polyribosome?
/polysome.
| two or more ribosomes translating one mRNA strand.
242
What happens to the secretory protein after moved into the Smooth ER?
lipids are made, so lipid modifications and vesicles formed
243
What happens to the secretory protein after moved to the golgi?
Addition of sugars and sorting takes place as moves from cis to trans.
244
Coatings of proteins as move?
Er to Golgi COPII
Through golgi COPI
secretion Clathrin
245
How are hormones/enzymes activated into mature form after synthesis?
Proteases cleave
246
Regulated vs unregulated vesicle fusion to the membrane?
Regulated requires a signal e.g. NT or hormone to cause e.g. Ca making Ach release
247
Signal sequence to the nucleus?
Lys-lys-lys-Arg-lys
248
What allows the attatchment of ribosomes to the translocator?
The SRP not only binds to a signal but creates a hinge around the large ribosomal subunit, binding these three together, as well as to the SRP Receptor on the ER which associates with the translocator.
249
How is it ensured that as the protein is translated it goes through the translocator?
The translation is paused as the SRP binds until the translator is opened.
The SRP and R are recycled.
250
What cleaves off the Signal recognition sequence?
Signal pepsidase
251
How can heavy and light chain vesicles be separated?
Differential centrifugation-
1. Homogenize the ER fragments into microsomes
2. Differential centrifugation to separate R from S microsomes from the ER
3.Use a tube with differential gradient of sucrose soltuion increasing down.
4 Centrifuge with the fragments.
5. Smooth low density float at low sucrose solution, and rough floats at higher sucrose so further down the tube.
252
How is transmembrane-ness of a protein achieved?
Has the normal start transfer sequence, but has a stop transfer sequence so it halts here through the transporter and when this dissociates this is left in the membrane.
253
How is membrane associated proteins achieved?
The signal can be swapped for a lipid anchor in the ER e.g. GPI anchor
254
Maturation of proteins?
Conformational maturation in ER after signal removed.
e. g Disulphide bridges are formed between cystine residues.
e. g. Glycosylation by a carbohydrate chain e.g. Oligosaccharides.
255
What do ER proteins have that ensures they stay in the ER?
A KDEL Amino acid sequence on C terminus, act as a localisation signal, either stop secretion or ensure their return.
256
Functions of carbohydrate modifications to proteins? (4)
Protein stability- protect
Cell-cell recognition and signalling
Cross species separation- recognition as foreign
Quality control mechanisms in the ER
257
example of foregin recognition by the carbohydrate modifications on proteins?
Humans use Beta-galatosidase and other animals use alpha.
258
How do carbohydrate modifications act as quality control mechanism?
Carbohydrate addition Via asparagine A.A.
Give info on structure, age, localisation of proteins.
Added in the ER
259
Structure of the quality control signal?
Glucose- Mannose- N-acetylglucosamine to asparagine
260
Modification of carbohydrate chains of proteins happens where?
In the golgi cisternae.
261
Examples of the editing in the golgi cisternae of carbohydrate chains from what structure to?
From the quality control sequence (glucose-mannose- N-acetylglucosamine)
glucose removed completely so:
N-acetylglucosamine to Mannose to N-Acetylglucosamine to galactose to NANA in 3 branches off.
262
Limitations od organ transplantation from other animals why?
However overcome?
Han cells B galactosidase whereas other animals use alpha- antibodies to and rejection.
Can be engineered to lack the alpha-galactosidase k/o enzyme that normally adds it. (alpha 1,3 galactosidase)
263
What do the carbohydrates also designate in the humans?
Blood type.
| The sugars on RBC's band 3 protein (an anion exchanger on the plasma membrane)
264
What are the blood types?
```
Depend upon your galactose.
O- no terminal galactose (nO)
B- just galactose
A- Acetylated galactose (A for acetylated)
AB- both
```
265
Which is the best blood to give to avoid rejection? Have? WHy?
O- If have no galactose at all will not give any antibody reaction. However would be bad to have as could only have O to you, as either A, B or AB will give a reaction.
THe best to have would be AB, as could then be given any of the blood types with no reaction.
266
What can blood types be used for as well as ensuring correct transfusions?
```
Paternity testing ruling out.
e.g. if O x O can only be O
whereas A x B can be any
B x B only O or B
AB x O only give A or B
Think if have double letter cant be O e.g. like crosses, and if no second letter will be O.
```
267
After insulin has been glycolylated what needs to happen before secretion?
Cleavage of signal sequence (pre-proinsulin in the ER) then C-peptide cleaved to be insulin by specific proteases in secretory vesicles.
268
Misfolding of Pro-insulin?
In the ER due to a mutation = Type 1 diabetes.
Protease cannot cleave off c-peptide.
ANtibodies made to these islets of langerhan cells.
269
How can cells process the same polypeptide into different hormones examples?
Opiomelanocortin can give ACTH or Lipotropin from the pituitary, and B endorphin by neurons in response to exercise and stress.
270
What is leprechaunism?
Insulin receptor defect.
Usually fatal within the first 2 years of life, elfin like facial features.
decrease in subcutaneous fat and muscle mass, increased hair growth on skin.
271
Rabson-Mendenhall syndrome?
Insulin receptor defect- survive to 20's.
| Skin aand teeth abnormalities, overgrowth in hair and pineal hyperplasia.
272
insulin receptor mutation diseases?
Leprechaunism, Rabson-Mendenhall
| Insulin resistance- type two diabetes
273
Where is insulin produced?
Beta cells in the islets of langerhans of the pancreas.
274
What keeps the two ends of the insulin peptide together after cleavage?
disulphide bridges between cysteines, after pro-insulin is cleaved- the centre bit.
275
What are the two enzymes that cleave pro-insulin?
Carboxypeptidase towards the Nterminal end, and Endoprotease at the C end (but both in the centre of the peptide to remove a section)
276
Short term function of insulin?
Glucose uptake from the blood and conversion to glycogen, for storage in muscles or adipose tissue.
1min
277
Long term function of insulin?
Increased expression of liver enzymes that synthesise glycogen (glyconeogenesis) and synthesis of ezymes that synthesise tricycloglycerols for storage.
around 12 hrs response, due to transcriptional regulation, but effects for weeks
278
Insulin receptor structure?
Receptor tyrosine kinasee
alpha and beta subunits synthesised as a single polypeptide but cleaved and held together by disulphide bridges. Two of these come together also held by disulphide bridge (dimer).
279
Ras protein kinase signalling steps in Insulin?
1. Insulin binds to RTK and causes autophosphorylation.
2. The RTK phosphorylated sites are recognised by insulin receptor substrate (IRS) which binds via a PTB domain, and is phosphorylated by RTK.
3. GRB2 binds to IRS via a SH2 domain
4. GRB2 interracts to Ras initiating the pathway.
5. Ras pathway involves the phosphorylation of MAP kinases
6. Eventually TF's are activated or inactivate, leading to change in gene expression.
280
What is a disulphide bond?
Oxidation reaction between two cystines.
281
PTB?
Phospotyrosine binding domain- binds proteins to Phosphorylated proteins.
282
Ras independent signalling of insulin?
1. Insulin binds and causes the autophosphorylation of RTK.
2. IRS (insulin receptor substrate) binds to this via a PTB group, and IRS is phosphorylated by RTK.
3. PI-3 kinase binds to IRS after Phosphorylation via a SH2 domain on the p85 subunit.
4. The p110 kinase subunit of PI-3 phosphorylates PI4-5bisphosphate to PI 3.4,5 Triphosphate and PI 4-phosphate to PI 3,4 bisphosphate.
5. This creates a docking site for PKB (Protein kinase B) on the membrane.
6. PKB is P by membrane associated kinases eg. PKD1, activating it.
7. PKB when active P glycogen synthase kinase 3 (GSK3) inactivating it, causing activation of glycogen synthase.
8. Glucose transporter GLUT4 translocation to the cell membrane so can bring glucose in from the cell.
9. FOXO is phosphorylated by PKB inacitvating it, therefore inactivating glucose synthesis by PEPCK.
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Ras independent signalling two subunits of PI-3?
p85- binds to IRSvia a SH2 domain
p110 kinase- phosphorylates PI 4-5 bisP to PI 3,4,5 TriP
and PI 4- P to PI 3,4 BisP
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P110 kinase allows what?
phosphorylates PI 4-5 bisP to PI 3,4,5 TriP
and PI 4- P to PI 3,4 BisP
Creates a docking site on the membrane for protein kinase B, which is then activated by phosphorylation by PKD1 (membrane associated kinase)
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Protein kinase B in Ras independent signalling does what?
- P GSK3 to inactivate it (which P glycogen synthase inactivating it so actually this activates it)
- FOXO P inactivating it therefore inactivating glucose synthesis by PEPCK
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What does FOXO do?
activates PEPCK which causes glucose synthesis
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What does GSK3 do?
phosphorylates glycogen synthase inactivating it
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PCR used for?
Polymerase chain reaction.
| Amplifying small amounts of DNA.
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PCR method?
1. Separate two strands by heating to 95 degrees.
2. Annealing of DNA oligonucleotide primers at much cooler temperatures (known sequence complimentary to at opposite ends of two strands)
3. Elongation of DNA using oligonucleotides added using a thermo-stable DNA polymerase e.g. Taq polymerase at 72degrees.
4. Repeated again.
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Other version of PCR?
Reverse transcriptase PCR.
1. DNA primer added mRNA reverse transcriptased into cDNA using deoxyribo-nucleoside triphosphates.
2. Separate strands.
3. Add second DNA primers and amplified by PCR into cDNA clones.
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What is promoter bashing?
Make a transgene that uses a quantifiable reporter e.g. luciferase which produces a luminescent substrate that is quantifiable, so when the promoter is active it makes the tissue emit light. Can then manipulate the promoter sequence (e.g. DNase 1 restriction enzyme) etc so see the effect on the fluroescence and therefore the gene transcription.
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How can promoter bashing be used example?
If make a series of deletions in the promoter by restrictive enzymes, can test the responsiveness to insulin of the receptor to find the critical region of the promoter which is critical for the enzymes function.
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Defective Nucleotide exision repair mechinery results in?
Xeroderma Pigmentosum (XP) and v sensitive to UV induced skin cancer.
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Nucleotide exision repair genes?
XP genes- XPA, C,D,F,G
Encode proteins that take part in the pathway
Homologues of ecoli Uvr proteins
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Genes encoding genetic recombinase repair mechanisms and cancer?
BRCA 2: breast, ovarian and prostate (repair damaged DNA)
ATM: Ataxia Telangiectasia- lymphoma/leukaemia risk
Fanconi Anaemia: FANC genes- leukaemia risk
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What is synthetic lethality?
When cells can no longer make repairs to their damaged DNA and die. If say both Homologous recombination and Base exision repair are faulty.
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H1?
Straps the DNA around histone octomers and stabilises it.
