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Cells > Semester 1 > Flashcards

Semester 1 Flashcards

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1
Q

DNA vs RNA

7

A
  • Both have phosphate backbones
  • RNA is single
  • RNA has a ribose with OH
  • RNA has uracil instead of thymine
  • RNA has a more complex structure due to folding on itself
  • RNA is very unstable, DNA can last many years
  • RNA can have non normal pairing of U and G
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2
Q

What can form when RNA folds on itself ?

A

Stem loop structures

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3
Q

How many types of RNA polymerase make RNA ?

A

Three

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4
Q

Transcription vs DNA replication

5

A
  • many RNA polymerase act on one DNA strand and make many mRNAs
  • the longer strands show where synthesis started
  • DNA replication has one DNA polymerase and makes one replicate strand
  • DNA replication needs a primer
  • two strands are templates in DNA replication and there is one template strand in transcription
  • higher error rate in transcription
  • the product of transcription does not stay bound to the template
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5
Q

RNA polymerase

3

A

Moves from 3’ to 5’

Template is antisense

Product is sense

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6
Q

Topioisomerases

A
  • RNA polymerase unwinds the DNA and pushed the coils up to one end creating super coils
  • unwinding ten base pairs is the same as removing one coil and will create an extra coil further up
  • topioisomerases cut one of the strands to release the coils and allow the progression of transcription and then reseal.

Type 1- one strand nicked and no ATP
type 2- nick both strands require ATP

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7
Q

TATA box

A
  • found 30 bases before the starting site
  • it tells the RNA polymerase where to start and is a general transcription factor because it is required to start transcription in all cells
  • the TATA binding protein binds to the TATA box and creates a kink before the starting site to allow RNA polymerase to bind
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8
Q

Large protein complex required for transcription to begin contains

A

RNA polymerase

Transcription factors

Mediators

Chromatin remodelling complexes

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9
Q

Splicing

A
  • removes introns
  • only happens in eukaryotes
  • can happen in more than one way
  • the sites for splicing have a G on each end
  • the branch site in the intron has an A which will attack the donor G splice site and break the phosphate backbone
  • the intron is separates from the exon
  • the A binds to the G and forms a lariat loop of RNA
  • the G at the other end attacks the loop and breaks it and separates the intron from the other exon
  • leaving a looped intron separated
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10
Q

Spliceosome

A

Enzyme required for splicing

5 small nuclear RNAs - U1 U2 U4 U5 U6

They recognise the sites for the spliceosome
They bend the RNA to bring the sites together
They catalyse the RNA cleavage

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11
Q

5’ RNA cap

A

For stability and binding to ribosomes

Added very early on

5’ to 5’ linkage of methylised guanine in the cap to the rest of the strand

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12
Q

PolyA tail 3’

A

For stability and helps the nuclear export of the mRNA

  • the end of the gene to be transcribed is signalled by a polyA sequence AAUAAA
  • the polyA binding proteins wait on the RNA polymerase until the end of the sequence
  • they wait there because the C terminal of the enzyme is phosphorylated like RNA backbone so they are attracted to it
  • at the end of the sequence the proteins bind and csf cleaves the RNA off the DNA template
  • cpsf stays attached to the RNA and recruits polyA polymerase
  • this adds up to 200 As and polyA binding proteins are then able to bind to the tail
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13
Q

Genetic vs epigenetic

A
  • both alter gene expression
  • genetic are permanent and are passed into somatic cells and the germ line
  • epigenetic sit on top of the sequence and don’t alter it
  • epigenetic can be reversed and are erased in the germ line

Waddington and the journey through the epigenetic landscape

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14
Q

Chromatin nucleosomes

A

The protein subunit of the nucleosomes are core histones

  • N terminal lysine rich tails on histones can be covalently modified
  • the tail has 30 amino acids and these are the first 30 in the histone components
  • 146 bases wound twice around a core which has eight subunits
  • there are two types of each of the four subunits and it’s an octamer
  • nucleosomes are electron dense and positive
  • condensed is innactive
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15
Q

Addition of acetyl and methyl and enzymes

A

Acetyl is COCH3
mono do or tri methyls added to lysine
They are competing reactions

Histone acetyltransferases add acetyl
Histone deacetylases remove acetyl
Histone methyltransferases add methyl
Histone demethylases remove methyl

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16
Q

Are Acetyl and methyl groups on active or innactive genes ?

A

Acetylation markers are found on active genes
It creates binding sites for activator transcription factors that contain a bromodomain. They also recruit TFs

Methylation markets are on both active and inactive genes.
Lysine sites 4 and 17 are active and create binding sites for TFs with zinc finger domain

Sites 9 and 27 are inactive. And creates binding sites for TFs with a chromodomain

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17
Q

Meta gene analysis

A

See if acetylation is correlated with the levels of gene activation.

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18
Q

Transcription activator proteins 5

A

Bind to the DNA sequence and recruit transcription initiation machinery

Relax the chromatin and make it more accessible

Remove nucleosomes to reveal the DNA

introduce unusual histone to facilitate DNA repair

Recruit enzymes to add or remove methyl or acetyl.

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19
Q

Transcription repressor proteins 3

A

Mask the activation surface so the activators can’t function.

Recruit chromatin remodelling complexes to condense the chromatin.

Recruit enzymes to remove acetyl groups.

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20
Q

Poly comb group

A

Two complexes

Genes that constrain each hox gene to a domain. The mutant is all hox genes expressed in all segments.

They encode proteins that chromatin repression so that the hox genes are restricted.

Enhancer of zeste is a histone methyltransferase which modifies lysine 9 and 27 and inactivates hox gene expression. The second complex.

It also encoded another protein that has a chromodomain which binds to the methyls added by zeste. This is the first part of the complex.

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21
Q

DNA methylation

A

Both DNA and histones can be methylised.
DNA methyl transferases add a methyl to cytosine to make 5 methyl cytosine which can recruit transcriptional repressors

Histone and DNA methyltransferases reinforce each other’s activity and both involve the polycomb group.

Inactive promoters are rich in methylated cytosine. It will recruit the repressor Me CP2 which brings deacetylases and methyltransferases which stop gene expression

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22
Q

Mammalian X chromosome inactivation

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A

One of the X chromosomes is selected for inactivation
Calico cat is heterozygous for fur colour
One of the chromosomes in each cell will be randomly turned off

XB or Xb is remaining in each cell causing patches of different fur.
Face neck and belly is white

Synthesis of a non coding RNA which spreads across a chromosome and shuts it down. The poly comb complex is recruited to add methyl groups and inactivate the chromosome.

23
Q

Barr body

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A

It becomes a Barr body which is a highly condensed inactive chromosome at the periphery if the nucleus.

24
Q

Agouti mouse

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A

Fur pigment and energy balance

Mutant is agouti viable yellow causes obesity and yellow fur

Dominant autosomal

Insertion of a retrotransposon which causes over activation of the gene

Feeding mothers high methyl diets can produce wild type offspring. The mutant gene will be repressed by methyl.

25
1944 Avery 1953 Watson 1966 Nuremberg
DNA carries genes DNA structure Genetic code
26
Non overlapping Degenerate Three reading frames
Read one codon then the next. Stops restriction of possible amino acid sequences. Many codons code for one amino acid. Depends on which base you start reading from.
27
Start codon Stop codon Open reading frame
Methionine AUG three stop codons UAA UAG UGA Stretch of nucleotides between start and stop
28
What serves directly as the template for amino acids
TRNA
29
TRNA
3’ end has amino acid Opposite not 5’ has anticodon Folded single strand Some bases are modified pseudouridine and dyhydrouridine
30
Wobble base
Allows the tRNA to bind to multiple codons The first two bind correctly The third can bind to two possible bases The RNA is distorted and bent and adenine can be Deaminated to make inosine which can being to U C or A.
31
Coupling of amino acid to tRNA
Amino acyl tRNA synthetase Adds AMP to amino acid so it is adenylated Creating a High energy phosphate bond on the amino acid The adenylated amino acid binds to trna and become aminoacyl tRNA or charged tRNA
32
Peptidyltransferase
In the ribosome Adds amino acids to the peptide chain High energy bond is broken and a low energy bond is formed on the chain
33
Protein structure
- simple backbone chain of C and N - the linear chain primary structure is held by strong covalent bonds - the backbone is the same for each protein but the amino acid side chains vary - the side chains are organised into secondary structure - the backbone is organised into a spiral and held by weak hydrogen bonds which are constantly breaking and reforming. - the entire structure is tertiary and has many helixes - proteins interacting with other proteins is quarternary
34
Protein folding
Polar side chains are hydrophilic Non polar side chains are hydrophobic Hydrophobic are hidden in the core and hydrophilic are on the outside forming hydrogen bonds with water.
35
Four ways a structure can be represented
Backbone Sticks Space filling Ribbon
36
src tyrosine kinase
Multi domain protein Involved in cancer Four domains. If one is removed and replaced the protein will still function
37
Finding primary structure 3 ways
Predict it using the DNA sequence Mass spectrometry Edman depredation- amino acid sequencing up to 60. Each round learns the identity of an amino acid. Pitch attached to the end of a protein and is acidified so an amino acid drops off. The amino acid is then analysed by liquid chromatography.
38
Finding secondary structure
Can be predicted from primary structure by biophysical modelling on software to find the lowest energy folding formation. De novo Can’t be wholly relied on.
39
Circular dichroism
Predict secondary structure Purified spectroscopy in the far UV region with short wavelengths Different structures absorb the light different amounts. The graph will show how much is helix and how much is sheet. Tertiary It can signal aromatic acids and disulphide bonds We can use temperature to follow the protein unfolding as it denatured
40
X day crystallography
Pure sample is concentrated to a crystal Shine x rays and see how the light is refracted Pattern can be used to calculate structure
41
NMR
- most atoms have a spin due to structure imbalance - wobble caused by uneven protons and neutrons - the wobble can be detected as vibration - label the protein with radioactive isotope - can count the C H N - can calculate structure - change the environment so the proteins resonate at different frequencies
42
Electron microscopy
Only able to see very large structures. Can’t be used for primary. Can see actin filaments like rope. Negative stain- protein in a slide is sprayed and the shadow left behind can create a structure.
43
How many proteins do cells contain In saliva In blood plasma
20,000 to 30,000 2290 2698
44
Protein purification basic
- homogenisation. Sonication vibrations. Blending - centrifugation - column chromatography. Sorted into size affinity and charge. To concentrate the protein. - confirmation by electrophoresis, western immunoblotting and mass spectroscopy
45
Centrifugation when purifying proteins.
After homogenisation Low speed- whole cells, nuclei and cytoskeleton Medium speed of supernatant- mitochondria , peroxiding sand lysosomes Fast- microsomes and small vesicles Very fast- ribosomes viruses and large macromolecule Leftover is pure cytosol The samples are tested for the desired protein to learn which organelle the protein is in.
46
Density based ultra centrifugation
- sucrose gradients are used - one round of centrifugation - the sucrose will be concentrated in the pellet - the structures are separated depending on mass and the more dense ones are found in the higher sucrose concentrations. - two substances being separated will be seen as two separate bands - empty the tube from the bottom and separate the layers into different tubes and collect different fractions.
47
Column chromatography for size
- beads have holes of a highly controlled size - larger molecules in sample can’t fit and have to go round the outside and reach the bottom fast - smaller molecules are slowed down by the beads. - buffer is added after the sample - the large molecules will go in the first fraction - the buffer will overtake the small molecules and be in the next fraction - the last fraction will be the small molecules - the machine swaps tubes at time intervals to get many fractions.
48
Affinity chromatography
- the beads have a covalently attached substrate - the enzyme in the sample will stick to the substrate and the other molecules will not - keep making fractions until nothing comes out and you know all the enzyme is bound - add an eluting solution to break the bonds as it’s a competing ligand - or add extra substrate so the enzyme binds to that and gets washed out - check the protein that leaves to make sure it’s the one of interest - on the graph each peak will show a fraction collected and the one with the desired protein is the one after the eluting solution is added.
49
Ion exchange chromatography
- separates based on charge - pos DEAE beads - neg CM beads - negative proteins bind to positive beads and the positive proteins can be flushed out - the bound ones are eluted by increasing the salt concentration so there is competition for the negative beads. - the graph shows the early cation peaks and the late anion peaks after there is an increase in salt. The last protein to leave is that with the most negative charge because it takes a higher salt concentration to remove it. Measure the fractions to find the one with the desired enzymatic activity.
50
3 step protein purification
Size exclusion Affinity Ion exchange To make sure the protein is pure
51
Gel electrophoresis SDS Beta metacarpa Problems
Proteins or DNA Protein- a gel box has two chambers containing buffer. The upper chamber has a negative current and the lower chamber has a positive current. A sample is put in wells and they are separated accordingly to size. Proteins are measured in daltons Proteins that are folded or modified could stick together and bias the sorting. SDS is used to unfold the proteins and give the uniform negative charges. Smaller proteins have less of a negative charge. Beta marcaptoethanol breaks the disulphide bridges to help unfolding Proteins are boiled for two minutes to denature them.
52
2D gel electrophoresis
Separated based on charge and size Fingerprint of the sample The graph shows the pH/charge against the size of the protein. The sample is added to the gel which has a pH gradient. The protein moves through the gel and stops when they are neutral with the pH After. The plate is turned 90 degrees and SDS is added and they are sorted into size.
53
Western blotting
This is done after 2D electrophoresis to detect individual types of protein using antibodies. Immobilise the proteins to make them available to the antibody. The gel is pressed against a membrane and a current is applied to migrate the proteins from the gel to the membrane The antibodies will bind and can be detected by light or colour change
54
Mass spectrometry
Determine protein structure Start with a pure sample Add trypsin a protease that chops the protein into peptides. The peptides are separated into individuals and ionisation is used to split them into single atoms This can be analysed by mass spectrometry to learn the total mass and charge of the atoms. We can predict the exact makeup of each peptide because there is only 20 amino acids and the peptides are small. The peaks on the graph are compared to other structures to identify the protein

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