1
Q
How do changes in gene expression underlie the diversity of cell types in the body?
A
1.All cells have same content but different set of proteins
diversity from gene expression
2.change expression due to signals and cues in environment
3.Cells express a fraction of their genes
4.express different genes when in disease state
2
Q
What affects the levels of expression?
A
The level of transcription
Less transcription= fewer expressed
3
Q
Where do positively charged binding DNA proteins reach?
A
They reach the into the major groove
+ve (R +L aminoacids) allow the protein to stick to the negatively charged phosphate backbone
4
Q
What is a DNA binding site?
A
Stretch of DNA unlike proteins, DNA has a limited topology
5
Q
How do TF recognise short stretches of DNA?
A
Through interactions with individual base pairs
H bond in the major groove
6
Q
Example of binding to DNA
A
Rox1 is known to bind 8 sites in yeast genes
Different sites have different affinity for the protein- consensus and frequency at each position
7
Q
Helix-turn-helix DNA binding motif
A
The recognition helix inserts into the major groove to make specific contacts
helix turn helix proteins tend to bind as dimers to 2 consecutive major groove
DNA recognition site is palindromic
8
Q
Zinc finger DNA binding motif
A
4 aa hold the zinc atom
An alpha helix recognises 2 bases
9
Q
Leucine zipper DNA binding motif
A
The alpha helical monomers are held together by hydrophobic aa
Homodimers bind symmetrical sequences
Heterodrimers bind non-identical sequences
10
Q
How is helix loop helix related to the leucine zipper?
A
Loop enables more felxibility
many DNA binding proteins bind as dimers cooperatively dramatically increases binding strength
TF are molecular- DNA binding domain, protein binding domain, regulatory domain, activation domain
11
Q
DNAse I footprinting
A
- radioactively label one end of the DNA
- Mix with cell extract (or purified protein)
- Add DNAse to partially digest the DNA
- Heat sample to destroy the DNAse ad release the binding proteins
- Run samples by gel electrophoresis
12
Q
What can DNA footprinting be used to identify?
A
Where a protein binds on a sequence of DNA
See the region of DNA protected from digestion by binding protein
13
Q
Electrophoretic mobility shift assay
A
- Radioactivity label of one end of the DNA
- Mix the cell extract (or purified protein)
- Run samples by gel electrophoresis
14
Q
What are the 3 forms transcription factors come in?
A
- Permissive- General TF are necessary for all transcription, bind at promoter
- Specific
- Regulatory
- activator= increase transcription of neighbouring genes
- repressor= reduce transcription of neighbouring genes
15
Q
How do regulatory transcription factors function?
A
Interactions with the RNA polyermase complex
Altering acetylation of the DNA
Binding to other transcription factors
Bind anywhere around the gene
16
Q
Why does DNA play an important role in forming DNA/protein complexes?
A
Chromatin does not easily bend so it is thought that for 2 proteins to interact they need to bind directly neighbouring
DNA sequences or to sites that are >500bp apart
17
Q
What are insulating barriers?
A
Block regulatory sequences from affecting neighbouring genes
18
Q
What are enhancers and silencers?
A
Enhancers= binding site for transcriptional activators Silencers= binding site for transcriptional repressors
19
Q
Genetic switch
A
Inputs that alter gene expression- strong inhitbiting protein, strongly activating assembly, weakly activating protein assembly
Each switch is responding to extrinsic or intrinsic regulation
eg. typtophan= repressor protein represses genes required to typtophan synthesis
20
Q
What are the many ways to regulate TF?
A
- Protein synthesis
- Ligand binding
- Protein phosphorylation
- Addition of 2nd subunit
- Unmasking
- Stimulation of nuclear entry
- Release from membrane
21
Q
Why do TF interact with each other?
A
Prevents them from falling off the DNA
Each protein needs to lose 2 interactions to fall off of the DNA
Binding of one TF to DNA may allow ANOTHER TF to bind to DNA
22
Q
Ways TF regulate the transcription of TF
A
- positive feedback
- negative feedback
- flip-flop device- inhibit each other (2)
- feedforward loop- miss one and activate the one ahead
23
Q
Key concepts
A
- gene control in the expression of precise decision so that the proper gene is expressed in the proper cell at the proper time
- The main mode of protein expression control is at the level of transcriptional initiation
- Regulatory sequence are numerous and diverse
- Transcriptional activators exert their effect either directly (recruiting components) or indirectly (modulating chromatin structure)
24
Q
How does alignment of genes show closely related species
A
Can identify conserved sequences of motifs
Often turns out to be the binding sites for regulatory proteins
Non-coding stretches of DNA change rapidly during evolution- coding sequence= conserved
25
TF interact with each other
1. Competitive DNA binding binding site for activator and repressor
2. Masking the activation surface
26
Activators are modular proteins, Example is
S.cerevisiae Gal1 gene- essential for the metabolism of galactose and is activated via the binding of Gal4 protein to the Vas regulatory sequence
*Upstream activating sequence
27
Epienetics
Study of changes caused by modification of gene expression rather than genetic code
Genetic alterations to DNA sequence can permanently affect gene expression whereas epigenetic changes to chromatin structure can also modulate gene expression but DO NOT alter the sequence and are REVERSIBLE
28
What is the difference between genetic inheritance and epigenetic inheritance?
Genetic inheritance- Gene x on- 1. DNA sequence change, turns gene x OFF- 2. multiplication of somatic cells- 3. production of germ cells- gene X still OFF
Epigenetic inheritance- Gene y on- 1. Chromatin change- gene y OFF- multiplication of somatic cells- 3. production of germ cells- Gene y ON
29
What do epigenetic modifications facilitate?
Stable changes to gene expression which may persist for life of cell/organism
Can be erased in germ line
30
What is an epigenetic landscape?
Different cell fates during development are the result of distinct journeys through this
31
What are nucleosomes?
a structural unit of a eukaryotic chromosome, consisting of a length of DNA coiled around a core of histones.
= Building blocks of chromatin
= covalently modified- these structural changes to chromatin affect gene transcription
32
How are covalent modifcations added to the core histones?
The N terminal lysine rich tails of core histones project radially from the nucleosomal core and are covalently modified
Variety of modifications added to the core within chromatin
33
The key modifications
1. Lysine actelyation
2. Lysine/arginine methylation- monomethyl, dimethyl and trimethyl lysine added to N terminals
3. phosphorylation
*Methylation and acetylation both occur on lysine-add lysine chains to lysine
34
Difference between HATs and HMTs
-Histone acetyltransferases
Can modify different lysine residues in core histones
-Histone myethltransferases
Exhibit exquiste site specificites- "Histone code" writers
Methylation and acteylation of some lysines are mutually exclusive (distinct reesidues mediated by different enzymes/regulationsand perception by cell machinery= selective)`
35
Effects on gene expression from Lysine/arginine methylation and acetlyation
H3-k4= active
H3-k9=Inactive
H3-k27=inactive
H3-R17=Active
Reversible- Histone dimethylases
36
Effects on gene expression from lysine acetylation
Many lysine in H2A,H3,H4= ALL active
37
What does the reversiblity of these modifcations mean?
That although relatively stable, if the correct signals are received then gene expression states can be changed
38
Acetylation of histones creates binding sites for transcriptional activation
Factors that contain bromodomain- an epigenetic code reader
| Mapping shows acetylation associated primarily with transcriptionally active promoter sequences
39
What can methylation of core histones create binding sites for?
Transcriptional repressors that contain a chromodomain
or
Transcriptional activators that contain a PHD zinc finger domain
Depends on the particular lysine amine acid residue modified methylation of core histones
40
Different histone modifications are distinct elements of transcriptional regulatory code
An epigenetic code that lies on top of the genetic code
| Governs when and where genetic info is exposed
41
How do transcription activator proteins work for chromatin?
- Selective nucleosome remodelling
- Selective histone removal- transcription is general TF mediator and RNA polyermase
- Selective histone replacement
- Selective histone modification- Recruitment of code writers and readers
Activator proteins induce combination of all these effects-promote RNA polyermase 2 recruitment
42
How doe transcription repressor proteins work in chromatin?
1. Competitive DNA binding
2. Masking the activation surface
3. Direct interaction with general TF
4. Recruitment of chromatin remodelling complexes
5. Recruitment of histone deactylases
6. Recruitment of histone methyl transferase histone deactylases (proteins that bind to methylated histones)
43
What is the polycomb group of proteins?
Polycomb repressive complexes, PRC
Include proteins that can generate or recognise regressive chromatin modifcations- A histone code writing or reading system
H3-k27 methylation mediated by enhancer of zeste (component of prc2)
44
2 Protein polycomb complexes
PRC2 - makes the mark - triggers transcriptional repression
| PRC1 - recognises the mark - maintains repressed state
45
Histone code reading and writing
H3-k27 methylation mediated by enhancer of zeste (component of prc2)(code writer)
Recruits Prc1 via polycomb chromodomain (code reader)
Formation of silent, repressed heterochromatin
46
Close relationships between transcriptionally repressive histone methylation and DNA methylation
Transcriptionally inactive promoters frequently rich inmethylated CPG
dinucleotudes- 5- methylcytosine
Addition of methyl groups to cytosine residues is mediated by DNA methyltransferase (DNMTs)
47
The histone methyltransferase E2H2 physically interacts with DNMTs
Together these enzymes mutually reinforce each others effects
48
How does inactiavtion of mammalian x-chromosome equalise the level of x chromosome derived products in males and females?
Males XY- 1 dose of x linked
Females XX- 2 doses of x linked
Cells in early embryo
Condensation of a randomly selected x chromosome
Direct inheritance of pattern of chromosome condensation
1. Only Xm active in the clone
2. Only Xp active in this clone
Only X chromosome is silenced in each somatic cell during early of female embryo Xp or Xm
Silencing= random
All progeny of each cell in which silencing decision was taken inherit same selected X chromosome ie Xp or Xm
49
The case of the calico cat for X chromosome inactivation
Males
- xOy
- xoy
Females
- xO xO
- xo xo
- xO xo
Calico cats exclusively female
Heterzoygous for 2 alleles of an x linked coat pigment gene- patches of orange and black because random x inactivation during early embryogenesis
50
Inactivation involves synthesis of what?
A non- coding RNA (Xist)from the X-iinactivation centre (Xic) on the chromosome destined for inactivation
-Xist bind to the X chromosome in cis and promotes chromatin condensation via a process that spreads away from the Xic in both directions
active- inactive
51
What does recruitment of modifying enzyme do to Xist RNA?
Recruitment to Xist RNA promotes formation of silent chromatin
recruits other polycomb group components
Leading to the H3K27 and H3K9 methylation of core histones in X chromosome chromatin
52
Sequence for activation
Active X + Xist RNA, PcG proteins, DNA methylation
leads to inactive X
Deactylation and hypermethylation of core histones and DNA methylation, leading to chromatin condensation and transcriptional repression
53
What do polycomb writers and readers detect?
Xist transcripts on the X chromosome
54
Barr Body
A highly condensed inactive x chromosome at the periphery of the nucleus of female somatic cells
A limiting amount of autosomal activator thought to maintain expression of the active X
55
From RNA to protein
mRNA carries the code from genome to ribosome
| the code translated by the ribsome into protein by tRNA the protein is folded into the appropriate shape by chaperones
56
History of the codon
1944-Avery provides evidence that DNA carries genetic info
| 1966- Nirenbery, ochoa and khorana elucidate the genetic code
57
4 key points about the genetic code
1. 3 bases encode aa- triplets
2. Non-overlapping
3. The code is degenerate, some aa are specified by more than 1 codon- 61 codons and 20aa
4. The code is read by a fixed starting point (AUG) and 3 possible stop codons ( UAA, UAG, UGA)
58
What is tRNA?
TransferRNA
Intermolecular base pairing within the tRNA gives it its structure
All tRNA have a similar structure- 4 leaf clover
One end base pairs with the codon=Anti-codon
Other end carries the aa- 3 end
59
How does the primary sequence of nucelotides vary within double stranded region modified by tRNA
Psuedourdine- T
Dihydrouidine- D
There are over 50 modifications that allow for more specific interactions with proteins
60
What is the ratio of tRNA to codon?
1:1 ratio
| Wobble bases allow the same anti-codon to bind to more than one codon
61
How is wobble made?
Modifications- deamination of A to create an inosine- i can pair to U C and A
bacteria use a few 3I tRNAs for the 61 codons
62
How is coupling of amino acids to tRNA formed?
Aminoacyl-tRNA synthestases
Synthetases first "primes" the amino acid by adding AMP to the c- terminus
It then uses adenylated aa to for the aminoacyl-tRNA
Aminoacyl-tRNA is charged tRNA because the energy of the ATP hydrolysis is still contained in the ester linkage (aa-O-ribose)
63
Translation of mRNA into precise amino acid requires 2 adapters:
1. The synthetase that pairs the correct amino acid to the correct tRNA
Aa have to fit into 2 different pockets in the synthetase (before and after AMP addition)
2. the tRNA that pairs the correct codon to the correct aa within the ribose
Pairing requires highly specific interactions between molecular sufaces
- linkage of aa to tRNA
- tRNA binds it codon in RNA
64
During protein synthesis new what are added to the c terminus of the protein?
Amino acids
| Peptidyl tRNA attached to c-terminas of the growing polypeptide chain
65
Protein synthesis takes part in the ribsome- What is included? and how does it work?
About 50 ribsomal proteins and several ribsomal RNAs (rRNA)
Divided into 2 parts (subunits)- the large subunit catalyses polymerization while small subunits facilitate the tRNA/mRNA interaction
Subunits come together on 5' end of mRNA- Process along the mRNA at 2 aa/second- then separate at stop codon
66
Translation review
1. Charged tRNA enters the ribsome at the A site
2. Peptidyl transferase catalyses amino acid addition, conformational changes move the tRNA to the E + P
3. Conformational changes move the small sub-unit 3 nucleotide
tRNA leaves E site
67
How do elongation factors help translation and improve accuracy?
Once the anticodon is bound- EF-1 causes 2 delays before the peptidyl transferase can act:
- First it must hydrolyse GTP to GDP
- Next it has to dissociate from the tRNA- checkpoint
2 pauses- both of these lags allow time for incorrectly bound tRNA to fall off
some of the correct tRNAs also fall off but at a slower rate
Hydrolysis of GTP occurs more rapidly is the codon and anticodon are matched correctly
68
What happens if synthesis is made in the absence of EF-1?
Then there are more errors in the protein synthesis
69
The ribosome is a ribozyme
RNA that catalyses a reaction
Large subunits rRNA form a massive structre that contains most of the catalytic activity including that of the peptidyl transferase
Riboproteins lie on the surface
70
Where does translation start?
AUG
The methiomine tRNA assembles the ribosome
Only the met tRNA with EIF-2 can bind to the small ribosome subunit alone
Complex^ binds to cap and tail is bound to the small ribosome EIF-4E to form the loop
71
What is EIF?
Eukaryote Initation factor
| There are many
72
Why do multiple ribosomes bind to mRNA?
Ribosomes are spaced out 80 nucleotides apart on a polysome
Stop codons are recognised by release factors, these look like charged tRNA and enter at site A- results in dissociation of the ribosome
73
When does folding of the protein occur?
Immediately after leaving the ribosome
As the protein exits it folds rapidly putting hydrophobic side chains in the middle to achieve a lower energy state
proteins initially fold into roughly the correct confirmation called MOLTON GLOBULE
Correct folding= multistep process
Incorrect state reduces energy state but blocks further folding= DEAD END
74
What do missfolding proteins generally have?
Exposed hydrophobic regions that can lead to aggregation
75
What are molecular chaperones?
Come to the rescue by reversing incorrect steps
Chaperone cataylsis- goes from OFF to ON pathway folding
2 major classes- Hsp60 and Hsp70
Heat shock proteins
Expression is elevated when temp is raised above normal
76
How does HSP70 work?
Works directly on protein as they exit the ribosome, binding to exposed hydrophobic amino acids
77
What do HSP60 do?
Put misfolded proteins into isolation
The hydrophobic entrance is unfoldeed
Use GroEs cap seals protein inside for 15 seconds to allow refolding and ATP + Pi
CORRECTLY FOLDED WITH HELP
78
Incorrectly folded proteins
Digested in proteosome- Pulyubiquintation marks translation failures for destruction in the proteosome
1/3 of newly synthesised proteins are immediately recycled
79
Diseases caused by protein aggregation
Large and protease resistant can lead to cell death
Chain reaction to missfold more
CJD- missfold proteins convert normal tissue
Huntington, alzheimers are all associated with large extracellular protein aggregates
Amyloid plaques- cross- beta filaments
80
Transcription
Process of RNA synthesis from a DNA template
DNA -1- RNA -2- Protein
1= transcription
2=translation
81
What are the 3 main types of RNA?
1. mRNA-codes for proteins (3-5%total for RNA)
2. tRNA- participates in translation (49 families) each carries an aminoacid and has a specific anticodon loop
3. rRNA- Ribsomal- 4 rRNA, constituent of ribosome
82
4 main differences between RNA and DNA?
RNA
1. Contain ribose (instead of deoxy-ribose)
2. contains U instead of T
3. Synthesised as single strand
4. RNA is very unstable
83
Secondary structure of RNA
```
Pairing between bases
- A=U
- C=G
Also can have non watson-chick pairing, such as G=U
formation of stem looping
```
84
Tertiary structure of RNA
Folding of molecule into 3 molecules
85
What are the transcription enzymes?
RNA Polyermase
- Enzyme performing RNA synthesis
1. RNA Polyermase 1- Ribsomal RNA
2. RNA polyermase 2- Protein encoding genes
3. RNA polyermase 3- tRNA, small nuclear RNA and 5 rRNA
86
Transcription requires RNA polymerase
Different from DNA replication
1. Multiple RNA pol bind on the same gene
2. no primer needed
3. only one strand of DNA is used as the template
4. Transcript does not remain bound to template
5. Higher error rate
87
How can genes be on either strand of the DNA
5'------3'
3'------5'
A) an RNA pol that moves Left to right make RNA by using the bottom strand as a template
b) RNA polyermase movind right to left is on the top
88
How does transcription create supercoiling?
DNA with fixed end
Unwind 10 DNA base pairs (one helical turn)
either get:
1. DNA helix must rotate one turn
2. DNA helix forms one supercoil
Topsoiomerases release supercoils to allow progression
89
Where does transcription for a gene start?
At its promoter
Promoter element for Pol2
Elements- Bre, TATA, INR, DPE
Sequences in the DNA tell RNA polymerases where to start
90
What is the protein complex required for transcription to start?
Enhancer- Binding site for activator protein
Activator Protein
Binding of general TF , RNA Polyermases mediators chromatin remodelling complexes and histone acetylases
91
How is RNA processed?
1. Splicing of intons- eliminates non coding region of mRNA to generate mature mRNA for protein synthesis
2. Capping of 5' end- necessary for stability, binding of mRNA to ribosomes and initiation of translation
92
How do we work with genes?
Standard DNA cloning:
- restriction enzymes
- Gel purification
- Ligation
- Vectors
- Transformation
Sources of DNA:
- cDNA library
- Genomic DNA library
DNA seqeuencing
93
1. How do restriction enzymes cut DNA into manageable sizes?
- act as dimers and recognise short palindromic DNA sequences
- Have precise recognition sequence
- some leave overhangs
- Others cut the DNA flush- In the middle
94
What is a blunt restriction enzyme?
Where it cuts the DNA flush- in the middle
95
2. How do restriction fragments get separated from each other by gel electrophoresis?
DNA is negatively charged- attracted to the anode which is positive
Gel= matrix- smaller fragments run further
Use dyes like Ethidium bromide to stain the DNA- purify the DNA from a small slice of gel
96
3. Cloning DNA involves ligation of 2 DNA fragments
Ligase is an enzyme that joins DNA fragments to create recombinant DNA
Cohesive termini (sticky ends) ligate because they can hybridize
-Xab1 cuts
-Spec1 cuts
Although the recognition sequences are different of Xab1 and Spec1 the overhangs are cohesive
97
4.Cloning DNA inivolves ligation of DNA fragments into a plasmid vector
1. Plasmids are small, circular extra-chromosomal DNA that occur naturally in bacteria
2. Have their own origin of replication that usually results in 50 copies of the plamid being made in each bacteria
3. Carry antibiotic resistance genes
4. Made by plasmids and adding a bunch of restiction enzymes in one part of the plasmid (multiple cloning sites)
5. Plasmids only hold <30 Kilobases of DNA
98
How much DNA do artifical chromosomes hold?
Bacteria artificial chromosome= <300 kilbobases
| Yeast artificial chromosome=<3 megabases
99
5. Cloning DNA involves transformation
Putting the DNA into bacteria
1. mix bacteria with plasmid DNA- creating temporary holes in the cell membrane
2. Competent bacteria= ready to take up new DNA- NOT very efficient process so treated bacteria selected on plates
3. Colonies grow that contain thousands of bacteria- each from single plasmid
4. Single colonies are lifted from plate to start a culture- plasmid easily purified
100
Where do we get the DNA to start with?
1. By making libraries of genetic clones= contains everything
- Advantage= contains all regulatory sequences, allowing one to study transcriptional regulation
2. By making libraries of cDNA clones from mRNA= only contains genes that are expressed
- Advantage= study diseases by identifying which genes are expressed in diseased tissue and compare with normal
101
How is cDNA cloned?
Cancer tissue- Extract RNA - Reverse transcription reaction - Ligate into a plasmid vector and transcription into bacteria (DNA copy) - Grow bacteria, culture and purify clone
When extract RNA from tissue a obtain population of 1000s of different RNA
102
What is a transcriptome?
The cDNA will represent genes that are expressed in the original tissue
103
cDNA library- isolating single clones from mixed population is important that there is only?
- One insert in each plasmid
- One plasmid in each bacteria
- One bacteria starts each colony
Some clones are house keeping genes and others tissue specific
Highly transcribed genes cloned more regularly, regulatory genes= more rare
104
Expressed sequence tags
Sequence the ends of all the clones in the library -genomic labs
105
Genomic library
Human cell - purify and digest chromosomal DNA with restriction enzyme- purify loads of different clones
106
What is a genomic sequence useful for?
Sequencing genes
107
Dideoxy terminator sequencing
To sequence:
1. Denature (100oc)
2. Allow to cool with primer
3. Start DNA synthesis reaction with DNA polymerase and DNTPs
108
What is the primer used for sequencing?
Oligomers- short 20nt, single stranded DNA that can easily be synthesised
Often designed to annneal to sequence on edge of vector
-DNA polymerase can't extend a strand a dppNTP has been added
-Add mix of Deoxy and deoxy nucleotides different strands will end at different positions
109
What does dideoxy terminator sequencing result in?
Separate bands on the gel
Running all 4 ddNTPs reactions on the same gel results in a nucleotide ladder
Sequence deduced from the binding pattern of autoradiogram made from gel
110
What is automated sequencing?
Up to 1000s of nucleotides read in one reaction
rather than labelled the primer, the ddNTPs are each labelled with a fluorescent dye
= simple reaction
While gel running, an electric camera takes pics of each band and measures its intensity (not whole gel just one position at a time)
111
How are the results of automated sequencing presented on a graph?
Intensity over time
| Graph= trace
112
What if DNA is longer than 1Kb (human 3.2^9)?
Pogressive sequencing= End of clones are sequenced using primers from the vectors. Primers are designed based upon the new sequence and another round of sequencing is performed
So on until sequence meet in the middle
113
BAC clones
Used to sequence a genome we sequence many BAC clones
| Can be localised to particular regions of genome and chosen for sequencing- needs planning and thought
114
Shotgun seqeuncing
Make a genomic plasmid library and just sequence the ends from each clone using primers from vectors
115
How are the short random sequences assembled into a contig? Advantages/ disadvantages
Computer programme
overlap
Ad- no thought= automated
Dis- need to sequence more than 6x the ste of genome to get large contigs- otherwise gaps
116
Both methods are parallel
Human genome
1. 1kd plasmid library- short- sequence end to produce 6 fold- assemble seq to chromosome
2. 100Kb BAC library- progressive- sequence entire BAC- assemble seq to chromosome
BMS238 Cell and molecular
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