BMS238 Cell and Molecular Biology Flashcards
Why is protein folding required?
For acquisition of the correct protein structure to perform designated function
What can protein misfolding lead to?
the failure to acquire the correct structure hence the failure of function
Give some examples of diseases that are caused by the protein misfoldings?
Huntingtons, Cystic fibroisis, Parkisons, Alzheimers
Why can researching protein structure be difficult?
Because proteins are made up of 20 different amino acids and the average protein has 500
- Each amino acid has its own complex structure e.g. alanine has 12 atoms
- So simplifying structural features allows easier understanding
What are the two main secondary structures of protein?
Alpha helix
Beta sheet
What are the different ways in which you can represent secondary protein structure?
- Backbone diagram
- Sticks (shows side chains)
- Space filling (shows all the atoms)
- Ribbon (follows the path of the backbone) - most useful
What is the structure of a beta sheet?
- Beta sheets consist of beta strands connected laterally on adjacent regions of the peptide backbone by hydrogen bonds. These interactions do not involve side chains
- The side chains extend above and below the beta strands allowing one side of the sheet to have different properties to the other
How are beta sheets usually represented?
As flat arrows pointing in the direction of the c terminus
- Central strands run parallel
- Peripheral strands run antiparallel
What is the structure of an alpha helix?
A spiral confirmation a N-H group from the backbone of an amino acid donates a hydrogen bond to the backbone C=O group of another amino acid located four residues further along the protein sequence. These interactions do not involve side chains
What amino acids do not favour alpha helix formation?
Proline and glycine
How has evolution fine tuned protein function?
By adding functional domains
What ‘interactions’ hold proteins together?
Hydrogen bonds
Electrostatic
Van der valls
Disulphide bonds
What are hydrogen bonds?
Involve a H shared between O and N atoms
What are electrostatic interactions?
Attraction between +ve and -ve charged ions
What are van der walls?
short-range hydrophobic interactions
What are disulphide bonds?
Involve a chemical link between two adjacent cysteines
What decides how a protein folds?
Hydrophobic and polar side chains
- Polar side chains form hydrogen bonds with water meaning they are on the outside of the protein to allow for reactions to occur
- Hydrophobic side chains are in the middle - repelling water forcing the protein to hold its shape
What are the levels of protein structure?
Primary - polypeptide chain
Secondary - alpha helix and beta sheets
Tertiary - whole protein
Quaternary - Several proteins
What is tertiary structure of a protein?
The way in which individual secondary structural elements; alpha-helices, beta-sheets and random coil, pack together within one protein
What is the quaternary structure of a protein?
Quaternary structure is the relationship between individual proteins in a multimeric complex (often duplication and multimersiation of subunits occurs).
Protein is said to contain subunits.
How do we find the primary structure of a protein?
Most primary structure is inferred from DNA sequence
But it can be determined directly by amino acid sequencing using Edman degradation or using mass spectrometry
What is Edman degradation?
- Phenyl isothiocyanate (PCT) is used to break away the terminal amino acid
- You can then determine the unique sequence by high performance liquid chromatography (HPLC). This is done based on the molecular weight of each residue
- Each amino acid has its own molecular weight, can therefore work out which amino acid is being removed each time and subsequently the primary structure
How can the primary structure of a protein be used to predict the secondary structure?
- By aligning it with other similar proteins (similar sequences of amino acids) with known structures
- Also some amino acids need to be in certain positions for structure to develop
What does amphipathic mean?
A polypeptide chain with both hydrophobic and polar side chains
How can alpha helices interact with each other?
Double or triple coiled coil
- Coil together alpha helices with their hydrophobic side chains facing inwards creating a hydrophobic core
Where are triple coiled coils found?
In elongated fibrous proteins that need the ability to form cloths
What techniques can be used to find real protein structure?
Circular Dichroism (CD)
X-ray Crystallography
Nuclear Magnetic Resonance (NMR)
Electron Microscopy
What is required for the techniques that are used to find real protein structure?
You need to purify the protein of interest
into its purest form.
What is Circular Dichroism used for?
To estimate the secondary structure of a protein?
How does circular dichroism (CD) work?
- CD spectroscopy uses far-UV radiation (190-250nm)
- Each secondary structure (alpha helix, beta sheet, random coil) give a characteristic shape on the CD spectrum. The fraction of the protein in each secondary structure can therefore be calculated from the CD spectrum, producing a percentage of each secondary structure present in the protein
- Α helices have two-peak spectrums at 208 and 225nm whereas, β sheets give a single peak between 216-218nm. Random coils also have a unique CD profile
How can circular dichroism be used to show protein stability?
Can be used at different temperatures providing us with information regarding the proteins stability – the less stable, the quicker the structure will be lost
What is calmodulin?
Calmodulin is a small calcium-binding protein which activates protein kinases and other proteins
Where are the binding sites on calmodulin?
There are two globular domains each with two calcium binding sites - total of four
Sites occur in the loop region connecting two alpha helices
What happens when 4 Ca2+ ions bind to calmodulin?
Allosteric changes
- The two globular domains would rotate allowing calmodulin to bind to target proteins and regulate their activity
- The central helix of calmodulin breaks into two allowing calmodulin to wrap around helical peptides on target proteins
What is NMR used for?
NMR spectroscopy is useful to understand dynamics of protein structures
What is requires of the protein of interest in NMR spectroscopy?
The protein of interest needs to be highly pure and labelled with isotopes such as 13C or 15N
How does NMR spectroscopy work?
- The subatomic particles of the isotopes possess a quantum-mechanical spin. These spin vectors are aligned with a large magnetic field in several configurations determined by energy state
- Radiowaves are then used to resonate with the natural frequency of these particles spins and cause a transition in spin vector orientation to a high-energy conformation.
- The NMR machine then records the different frequencies required for resonance to occur. This attribute, known as chemical shift, is dependent on the local environment and can be used to determine protein structure
How can you label a protein with an isotope (as required in NMR)?
The protein must be highly pure so is produced recombinantly in bacteria that have been grown on a media where the sole nutrient source is 13C or 15N. This will produce the protein labelled with these isotopes
What is x ray crystallography?
A high energy and focussed beam of X-rays is fired through a protein crystal. Most of the X-rays go straight through, but a few are deflected giving rise to a diffraction pattern. The structure of the protein is effectively traced back from the diffraction pattern using modern computational techniques
What are the two type of electron microscopy used to study protein structure?
Transmission electron microscopy
Cryo-Electron microscopy
What is transmission electron microscopy used for?
To study large protein structures
How is transmission electron microscopy used to study protein structure?
The protein of interest is placed on an EM grid and spiked with a solution containing a heavy metal shading that is impermeable to electrons
Electrons are then fired at the protein and a negative stain is produced
Image analysis is then employed to build-up an average structure
How is Cryo-Electron microscopy used to study protein structure?
- Cryo-EM consists of using liquid nitrogen to preserve/freeze the protein specimen
- Electrons are then shone onto the frozen specimen and the average shape of the protein is determined
- The more ordered and symmetrical the protein specimen is that is used in cryo-EM, the easier the averaging process
What are the pros and cons for using circular dichroism to study protein structure?
- Cheap, Quick, no size limit
- Limited dynamics, low resolution, secondary structure only
What are the pros and cons for using X ray crystallography to study protein structure?
- High resolution, no size limit
- Expensive, slows down due to crystal formation
What are the pros and cons for using NMR to study protein structure?
- Reasonable resolution, good dynamics
- Size limit of 50kDa
What are the pros and cons for using electron microscopy to study protein structure?
- No size limit - large structures
- Quite expensive, medium speed
- No dynamics
Why do we need to purify proteins?
Because cells contain 20000-30000 proteins
We therefore need to know the structure and function for each individual protein
Even secretary fluids have a large mix of proteins
Give the steps in protein purification
- Tissue homogenisation by sonication, blending, pestle and mortar
- Separation of the released material from unbroken material by centrifugation
- Several chromatography steps
- Confirmation of protein purity by electrophoresis
- Confirmation of protein identity by Western immunoblotting
- Confirmation of protein identity by mass-spectrometry
What are the steps in differential centrifugation?
Spin the cell homogenate at a low speed
- pellet containing whole cells, nuclei, cytoskeletons
Spin the supernatant at a medium stage
- Pellet contains mitochondria, lysosomes and peroxisomes
Spin at a high speed
- Pellet contains microsomes and small vesicles
Spin at a very high speed
- Contains ribosomes, viruses, large macromolecules
- Pure cytosol
What are the two types of density based ultracentrifugation?
Velocity sedimentation
Equilibrium sedimentation
What is velocity sedimentation ultracentrifugation?
- A test tube containing a stabilising gradual sucrose gradient is established (highest concentration at the bottom)
- Addition and centrifugation of the cytosol separates components based on density
- A hole is punctured in the bottom of the test tube and organelles can be collected from the bottom in various fractions depending on their density
- Heavier organelles will sediment quicker and lighter ones will take longer
What is equilibrium sedimentation ultracentrifugation?
- A steep sucrose gradient is established in a test tube with highest concentrations at the bottom
- The cell contents are then added and centrifuged for a long time at a very high speed
- The organelles deposit in the sucrose depending on their density with heavier, denser organelles depositing at the bottom of the tube and so on
What is gel filtration chromatography?
- Cytosol that has previously been centrifuged is loaded into a column containing a solid matrix of beads of a certain size
- Neutral buffer and solvent added to push cytosol down column
- Larger proteins have a smaller volume to move through, due to their size. Larger proteins leave the column first
- The different fractions collected drop-by-drop over time
How is the working range of the resin used in gel filtration chromatography defined?
The resin used has a working range which is defined by the pore size of the beads. If proteins are too large to fit through the pores, they are excluded
What is affinity chromatography?
Relies on the tight interactions of enzyme-substrate binding and allows separation of specific substrate binding proteins from the cytosol. Bound protein is eluted using a competing ligand or high salt concentrations
How do you purify specific DNA binding protein by affinity chromatography?
- Cytosol added to column containing beads with a covalently attached substrate
- Enzymes for the substrate bound to the bead bind irreversibly if the substrate is non-hydrolysable
- Other proteins in the cytosol will pass straight through the column and only the enzyme will be retained
- Elute the protein of interest using salt
What is ion exchange chromatography?
- Diethylaminoethyl (DEAE) beads added to a column in addition to negatively charged proteins
- The proteins will bind to the positively charged DEAE beads
- An increasing concentration of salt is then added to the column to displace the proteins form the beads
What solvents do you use in ion exchange chromatography?
Cation exchange
- CM carboyx-methyl
Anion exchange
- DEAE diethylaminoethyl
How do you remove the proteins from ion exchange chromatography column?
- Less negatively charged proteins are displaced and release from the column first, at lower salt concentrations
- More negatively charged proteins will be displaced later at higher salt concentrations. This allows you to separate fractions based on charge
What is meant by the three stem protein purification?
Using a mixture of ion exchange, gel filtration and affinity chromatography
What is SDS-PAGE?
Sodium dodecyl sulphate polyacrulamide gel electrophersis
Outline SDS-PAGE
- Proteins are boiled and SDS added. SDS is negatively charged and hydrophobic. Proteins fold hiding the hydrophobic centre. Proteins can then unfold after the addition of the hydrophobic SDS forming a stick allowing them to move easily through the gel. SDS makes the proteins have an equal charge to mass ratio
- Proteins are placed in a well behind the gel and a current is applied
- The negatively charged proteins move towards positive anode
- Smaller proteins will move faster and hence further through gel whereas larger proteins will move much slower
Why are proteins essentially sorted by molecular weight not charge in SDS-PAGE?
- SDS coats the proteins with a uniform negative charge, masking their charges
- SDS binds fairly uniformly to the linear proteins meaning that the charge of the protein is now approximately proportional to its molecular weight
- This means that there will be now differential migration based on charge so larger proteins will have the same attraction to the anode as smaller proteins but larger will travel slower as they can’t pass through the gel is easily
What is the role of mercaptoethanol?
Denatures the protein structure due to its CH group
Breaks down polypeptide chains into smaller fractions which can then be investigated separately
What is 2 dimensional gel electrophoresis?
Used for complex samples
1st dimension
- A stable pH gradient is created in a commercially available gel. Proteins are introduced to the gel and run along it until they reach the pH that corresponds to their isoelectric point
- At this pH, the proteins become uncharged and no longer run along the gel causing the proteins to separate by their native charge
- Commonly used in proteomics and in complex samples
What is western blotting?
- Separated proteins are transferred to a membrane using electrical current
- Excess of primary antibody is added to the membrane which binds to target protein
- After the unbound antibody is washed off, an excess of a secondary antibody is then applied to the membrane
- Secondary antibody binds selectively to the Fc region of the primary antibody
- It is tagged with an enzyme which when activated leads to the production of a fluorescent or coloured product
Why must we use two antibodies in western blotting?
It would be too expensive to label each primary antibody with an enzyme as there is too many
Instead use secondary antibody that can detect the primary ones
Secondary are only used to determine location of unlabelled primary antibody
How can you detect the antibody binding in western blotting?
H202 addition produces lots of light can look via X Ray
What is mass spectrometry?
Allows us to identify the labelled peptide
- Isolated protein incubated with protease (trypsin or peptitrypsin) to digest it into peptides
- The small peptides are then run in mass spectrometer which ionises the peptides - You can then identify specific peptides using its database of known peptides molecular weights to compare with
Why is trypsin used in mass spectrometry?
Because only small polypeptide chains can be put into the mass spectrometer
- Trypsin cleaves the polypeptide chain after lysine
- Lysine has an abidance of 6% in humans so is likely to result in a small chain
Trypsin is also cheap
Why is mass spectrometry useful?
Because it allows identification and sequencing of tiny changes in proteins
How does a molecule being labelled by a phosphate affect its mass spect result?
The presence of a P04 group increases the mass of serine by 95 daltons so a fraction will change if labelled with a phosphatase
Which amino acids can be phosphorylated?
Serine
Threonine
Trysoine
Because they have hydroxyl groups
Define proteomics
The term Proteomics refers to the analysis of complete protein content in a living system, including post-translationally modified proteins and alternatively spliced variants
Why is mass spectrometry crucial for proteomic studies?
Mass Spectrometry has now become a crucial technique for almost all proteomics experiments. It allows precise determination of the molecular mass of peptides as well as their sequences. This information can very well be used for protein identification, de novo sequencing, and identification of post-translational modifications.
Name some post translational modifications of amino acids
Hydroxylation
Methylation
Acetylation
Lipids
How does hydroxylation change an amino acid?
Adds an OH group
+17 Daltons
How does methylation change an amino acid?
Addition of CH3 group
+15 Daltons
Has an important role in epigenetics
How does acetylation change an amino acid?
Addition of CH2CH3 group
+27 Daltons
Role in epigenetics
How do lipids change an amino acid?
Varies depending on the modification
- Usually +2000 Daltons and higher
When were chromosomes discovered?
1902 by Boveri and Sutton
Morgan discovered that chromosomes are the locations of genes in 1915
What is contained in chromosomes?
DNA and proteins
What are the functions of the proteins in chromosomes?
- Packaging and unfolding of DNA within the nucleus
- Controlling DNA replication, DNA repair, genetic recombination
- Maintaining chromosome integrity by preventing loss of end sequences
- Governing proper chromosome segregation during cell division
- Regulating gene expression
Are chromosomes only found in the nucleus?
No small circular chromosomes are found in mitochondria and chloroplasts
At what stage in the cell cycle are chromosomes easily distinguished?
Metaphase
Define karyotype
The organised representation of all chromosomes in a eukaryotic cell and metaphase
How can different chromosomes be distinguished from each other?
Chromosome painting
- Allows chromosomes to be distinguished based on its DNA sequence content
- This technique is often used in place of measuring chromosomes
Measuring chromosomes
- Each pair of chromosomes are different sizes
How are chromosomes arranged in a nucleus undergoing interphase?
Normal running of cell - housekeeping functions
Can see that each chromosome occupies a distinct subnuclear territory in the interphase nucleus
How does the location of transcriptionally active and inert genes differ in a nucleus?
Active genes are in the centre and inactive are in the peripheral
There are some exceptions but this is generally the pattern
How was the location of active and inert genes investigated?
Gene specific paint was used to investigate the position of a signal responsive gene
- When active the genes (one on each chromosome) can be seen in the centre of the nucleus
- When inactive the genes were pushed towards the periphery
What is a chromosome?
A highly coiled fibre of chromatin
What is seen if you look at an interphase chromatin under an electron microscope?
Resembles beads on a string where the beads are nucleosomes
What is the structure of a chromatin fibre?
It is a supercoiled array of nucleosomes
What is a nucleosome?
DNA wrapped around a protein core
Describe the protein core of a nucleosome
Consists of 8 subunits called histones
- There are 4 types of histones, each with two copes
- The N terminal tails of the histones project out of the nucleosome and are free to interact with other proteins facilitating regulation of chromatin structure and function
What is the role of histone H1?
It is a linker histone that straps DNA onto histone octamers and its the movement if DNA relative to the histone octamer
- Important in chromatin condensation process which shrinks interphase chromosomes to metaphase size in cell division
What is the structure of the N terminal tail of histone molecules?
Rich in basic amino acids such as lysine
How can remodelling of chromosome structure occur?
Axillary proteins strip off DNA by removing histone H1
This allows the proteins to displace the histone core away from the DNA that is it is usually bound to
Produces a stretch of nucleosome free DNA which can be readily transcribed - accessible by transcription factors
Why do chromosomes need to contain specialised DNA sequences?
Because the sequences facilitate reliable and complete DNA replication, segregation of duplicated chromosomes during cell division
What is a telomere?
DNA sequences at ends of linear chromosomes: maintain chromosomal integrity
What is a replication origin?
DNA sequence where DNA replication is initiated
What is a centromere?
DNA sequences on which kinetochore assembles and mediates chromosome segregation at mitosis and meiosis
What is a kinetochore?
Protein complex that binds microtubules in the mitotic spindle
What is the role of a telomere?
A specialised DNA polymerase called telomerase produces a single stranded 3” overhang repeat called a telomere
- Repeat is TTAGGG
- Added after replication and stops the removal of DNA nucleotides at the end of the DNA strand
Where are centromere seen?
In the centre of the nucleus at the end of microtubule attachments
What do centromeres consist of?
Alpha satellite DNA repeats which form condensed heterochromatin with histone octamers containing unusual subunits
How does the kinetochore have a role in cell division?
Kinetochore inner plate proteins bind to alpha satellite DNA
Kinetochore outer plate proteins bind with microtubules (component of the mitotic spindle)
This arrangement occurs on both sister chromatids so that when division occurs the they are pulled in opposite directions
What is different about the structure of the nucleosome in centromere chromatins to normal DNA chromatins?
They have a centromere specific histone 3 variation called CENP-A that is only found in the centromere chromatin and is responsible for the physical interactions with the inner plate proteins in the kinetochore
What is the role of chromatin containing normal histone H3 in the centromere?
Ensure the right pairings of sister chromatins - allow them to be next to each other
How do kinetochore interactions differ in yeast to animals?
In yeast the kinetochore is a basket that links a single nucleosome of centromeric chromatin to a single microtubule
What is the eukaryotic genome made up of?
1.5% protein encoding
50% repeated DNA sequences
20% introns
30% are non of these but are still unique sequences
- Thought to be regulatory and control the activation of the protein encoding genes
What are the repeated DNA sequences of the eukaryotic genome made up of?
A few % DNA transposon
10% retroviral like elements
30% non retroviral polyA rectotransposons
All are genetically mobile and can relocate on the genome
What are DNA transposons?
occupy a few% of the eukaryotic genome
- DNA sequences that are bound by short repeated sequences
How are DNA transposons genetically mobile?
They move by relocation
- Uses transposases in a cut and paste mechanism
- Transposases bundle the transposon up and move it to another location
- The original location is then transposon free and the DNA strand is fixed
How discovered DNA transposons?
Babara Mcclintock - 1952
What are retroviral like elements?
Occupy 10% of eukaryotic genome
- Integrated DNA sequences that transcribe and produce proteins
- These proteins convert RNA copies of the retroviral genome back to double stranded DNA using reverse transcriptase
- This is then inserted back into the genome in a different location
What are non retroviral polyA retrotransponsons?
30% of eukaryotic genome
- Most abdudent type of transposons in the vertebrate genome
- Replicate via RNA intermediate
How do non retroviral polyA retrotransponsons genetically relocate?
Double stranded DNA sequence is transcribed and encoded proteins that have both a endonuclease function and reverse transcriptase function
This leads to the insertion of the newly reverse transcribed DNA into the genome at a different location as the DNA strands have been cleaved by the endonuclease function
What is the Alu gene?
Found in humans and evolved from a dingle gene called 7SL RNA but now has 1000000 copied of this gene
Through the process of non retroviral retrotransposition
What is the mouse homologue of Alu?
B1
How is DNA replicated?
Semi conservative replication
- Each strand is used to make a daughter strand
Which direction can phosphodiesterase binds form?
5’ to 3’
Define 5’ and 3’ end
5’ - Phosphate present on 5’ carbon of the deoxyribose sugar
3’ - OH present on the 3rd carbon
How is a RNA primer extended in DNA replication?
The OH group on the 3’ end carries out a nucleophilic attack on the phosphodiester bond on the incoming deoxyribonucleoside triphosphatase (e.g.cysteine)
- Also produces pyrophosphate
- This stabilises the reaction and makes it effectively irreversible as the breakdown of pyrophosphate by pyrophosphotase (breaks two high energy phosphate bonds) produces lots of energy that is used to form the new phosphodiester bonds
Why is DNA synthesis effectively irreversible?
Because the breakdown of pyrophosphate is extremely exothermic providing the energy needed for DNA synthesis - a coupled reaction
It is therefore effectively irreversible as it would take a lot of energy to be created to match the energy produced by this reaction
What is a replication fork?
Where the DNA strands are separated by DNA Helicase so that DNA synthesis can be initiated
What is the problems with DNA synthesis from the lagging strand?
Replication can only occur in the 5’ to the 3’ direction meaning that the lagging strand cannot continuously be synthesised as its in a 3’ to 5’ direction
How does synthesis of the lagging strand occur in DNA replication?
By synthesising discontinuous fractions called okazaki fragments
Requires contoinous RNA primers to keep synthesising small fragments
- There are short gaps between the Okazaki fragments
- Rionuclease H removes RNA primer creating the gap
- DNA polymerase extends across the gap and DNA ligase covalently links the fragments to make the stand continuous
What is the role of the RNA primer in DNA replication?
DNA primase synthesises a RNA primer from which DNA polymerase can extend from
What is the role of DNA ligase in DNA replication?
DNA Ligase uses the energy of ATP hydrolysis to ligate newly
synthesised, adjacent DNA fragments in a two-step catalytic reaction
- It uses ATP to create a structure where adenosine diphosphate is added to 5’ phosphate facilitating the nucleophilic attack of the 3’ OH of the Okazaki fragments
What is the role of DNA helicase in DNA replication?
DNA Helicase uses ATP to separate parental DNA strands at the
Replication Fork and move the Replication Fork forward
It wraps around and rotates the strand, disrupting the DNA
What can mutations in DNA helicases lead to?
Werner syndrome - Autosomal recessive - Mutation in RECQ helicase gene WRN - Progeria (premature ageing) Bloom syndrome - Loss of function in RECQ family of DNA helices which maintains genome integrity - Rare cancer syndrome
What is processivity in regards to DNA polymerase?
That DNA polymerase is more likely to extend DNA then it is to fall off
-This is greatly enhanced by its association with a sliding clamp which is positioned close to the primer:template junction
What is the sliding clamp?
The sliding clamp is ATP dependant
- The clamp loader and the sliding clamp create a structure that attracts and stabilises DNA polymerase
- It sits on the primer junction
- It looks like DNA helicase and it sits around DNA and spins to help DNA polymerase move forward
- The sliding clamp sits behind and the DNA helicase in front and DNA polymerase in the middle
What is the problem encountered when DNA helicase has separated DNA strands and then moves on?
The single stranded DNA may start to fold back on themselves causing them to become double stranded
How is the re joining of single stranded DNA strands in DNA replication overcome?
Using single stranded binding proteins
- They keep the DNA strands straight to stop them from folding back on its self and becoming doubled stranded
What is the role of DNA Topoisomerases in DNA replication?
DNA topoisomerases prevent DNA from becoming tangled during DNA replication - Unwinding of parental DNA strands at the Replication Fork introduces superhelical tension into the DNA Helix. - Tension is relaxed by DNA Topisomerases, which nick and reseal the backbone of the parental helix
WHat is the difference between type I and type II Topoisomerases?
- Type I Topoisomerases nick and reseal one of the 2 DNA strands, no ATP required - Type II Topoisomerases nick and reseal both DNA strands, ATP required
What is the role of replicators/orgins in DNA replication?
They are specific DNA sequences that direct the initiation of DNA replication by recruiting Replication Initiator proteins
Give an example of a replicator/origin for DNA replication in yeast
Autonomously Replicating Sequence (ARS) Elements
Give an example of a replicator/origin for DNA replication in humans
DNA sequences near to LMNB2, MYC, HBB.
But also defined by chromatin structure (e.g.
nucleosome free region), rather than a specific DNA sequence.
What is required for initiation of DNA replication in eukaryotes?
- Replicator Selection - formation of a pre-Replicative Complex (pre- RC) - occurs in G1 phase
- Origin Activation - unwinding of DNA and recruitment of DNA Polymerase - occurs in S phase
How is it ensured that each chromosomes is only replicated once per cell cycle?
Temporal separation of replicator selection and origin activation events ensures that each origin is used and each chromosome is only replicated exactly once per cell cycle
How is the pre replicative complex (pre-RC) formed?
Occurs in G1
- Origin recognition complex (ORC) binds to replicator sequence
- Activates helicase loading proteins Cdc6 and Cdt1 waging bind to the ORC
- The helicase Mcm2-7 binds as well completing the formation of the pre-RC
How is the activity of pre-RC regulated in DNA replication?
High levels of Cyclin-dependent kinase (Cdk) activity in s phase activates existing pre-RC complexes
Low levels of Cdk in G1 allows the formation of pre-RC formation
What would happen if there wasn’t any telomeres?
Removing RNA primer at the end of the lagging strand by ribonuclease H leaves a gap at the end that cannot be closed by DNA polymerase and ligase leasing to incompletely replicated DNA
If this wasn’t solved then it would lead to progressive shrinkage of chromosomes as ribonuclease H will keep removing RNA primer at 5’ end of lagging strand
How is the progressive shrinkage of DNA strands overcome in DNA replication?
Addition of TTAGGG repeats by telomerase compensates as the loss of telomere sequences caused by RNA primer removal end prevents chromosome shortening
The extended 3’ end DNA is long enough to enable DNA Primase to bind and initiate new RNA primer synthesis which can then be extended as an extra Okazaki fragment
How does telomerase produce a telomere sequence?
Telomerase is a ribonucleoprotein with an intrinsic RNA component that acts as a template on which telomere repeat sequences are synthesised in a step-wise process
– the Telomerase Shuffle
Outline the telomere shuffle
Telomerase extends the 3’ end of the DNA strand by 3 nucleotides, shuffles along and then disengages its RNA
The telomerase then moves along 6 nucleotides and drops down again (base paired with the newly synthesised bases). Then synthesises 6 new bases and shuffles along
This repeats
How do proteins interact with each other?
No chemical bonds are formed, just multiple weak interactions
What is the charge (usually) of a protein DNA binding domain?
They have an overall basic charge (positive) to mediate the interaction with the acidic (negatively charged) DNA strand mostly through interactions with the major groove
What are the classes of DNA binding domains in proteins?
- Leucine zipper motifs
- Zinc fingers
- Basic helix-loop-helix (bHLH),
- β-sheet
How do proteins increase affinity for DNA?
Increase the number of DNA binding motifs they have
What is a leucine zipper domain and how does it bind to DNA?
- Dimers of short coiled-coil sequence and a specific DNA recognition helix
- Two long alpha helices with hydrophobic side chains extending out into space between them (often leucine). These side chains are tightly packed adding stability to the domain
- The side chains extend into the DNA groove to contact bases forming Hydrogen bonds
Give examples of how metal is bound by proteins
Structurally - Zn2+ - Zinc finger
Regulatory - Ca2+ - Calmodulin
Catalytic e.g.. Zinc, iron and copper
What is Zinc finger DNA binding?
- Zinc finger proteins recognise specific DNA sequences
- Tetrahedral shaped by two cysteine residues from the alpha helix and two histidine from the beta sheet
- The helical region of each zing finger rests in the major groove of the DNA and the side chains project outwards, contacting bases
- The identity of these side chains determines the specific DNA sequence that is recognised by each finger allowing sequence specificity of the protein
Why are multiple zinc fingers used in DNA binding?
Because one zinc finger only interacts with two nucleotide interactions - not strong so use multiple zinc fingers
Outline DNA gel electrophoresis
DNA migrate from minus to plus end of the field. If DNA was interacting with a protein then the DNA will be shifted several positions depending on which protein they interact with
What is the purpose of the salt gradient in DNA gel electrophoresis?
Using a salt gradient you can investigate the strength of protein interaction with DNA
The strongest interacting proteins will need high salt concentrations and weakly interacting will require low salt concentrations
What is the purpose of DNA foot printing?
To confirm DNA binding
How is DNA foot printing carried out?
Label DNA with radioactivity and then digest the DNA nucleotide by nucleotide
If a protein is interacting with the DNA sequence then you will see that you’re missing some of the degradation products - can see that protein is bound
What interactions occur involving a zinc finger?
Binds zinc to DNA
What interactions occur involving a leucine zipper?
Protein - DNA binding
What interactions occur involving an EF hand?
Binds calcium or magnesium in structural or signalling mode
What interactions occur involving a SH2 domain?
Binds phosphorylated proteins
What interactions occur involving a SH3 domain?
Binds proline rich motifs
What interactions occur involving a PH domain?
Binds phosphorylated lipids
What DNA binding domains are involved in the insulin signalling pathway?
- SH2 domain - binds phosphorylated tyrosine (Grb2 protein)
- SH3 domain - binds proline-rich motifs (Grb2 protein)
- PH domain - binds phosphoinositide lipids (IRS1 and Sos proteins)
Outline the insulin signalling pathway focusing on the protein domain interactions
- The activated receptor phosphorylates itself on tyrosine - This then recruits a protein called insulin receptor substrate-1 (IRS1) via a PTB domain of IRS1
- the PH domain of IRS1 also binds to phosphoinositides on the plasma membrane
- the activated receptor phosphorylates IRS1 on tyrosines, and one phosphotyrosine binds
the SH2 domain of the adaptor protein Grb2 - Grb2 uses two SH3 domains to bind to a proline-rich region of a protein called Sos,
- Sos also binds to phosphoinositides in the plasma membrane via its PH domain.
- Grb2 uses its other SH3 domain to bind to a proline-rich sequence in a scaffold protein.
- The scaffold protein binds several other signaling proteins, and relays the signal further
Outline the characteristics of the SH2 binding domain?
It is an important phospho-tryosine binding domain often involved in signalling mechanisms
- important in the formation of signalling complexes
- Binding occurs between the negative phosphate group and the positive amino acid
- Hydrogen bonds also contributes
Where is the original SH2 binding domain found?
Protein tyrosine kinase Src
Outline the characteristics of the SH3 binding domain
Use aromatic amino acid stacking (hydrophobic staking) to bind its ligand
- It is a polypro line binding domain
- Involved in linking signalling components and maintaining multi protein complexes
- The minimum consensus sequence for SH3 binding are proline - two amino acids - Proline
- Tyrosines and tryptophanes – are positioned such that they
can stack with the aromatic rings of proline residues in target proteins
Outline the characteristics of the PH binding domain?
- Involved in membrane binding, signalling and anchoring proteins to membranes
- The PH domain interacts with charged head groups of phospholipids anchoring the protein to the membrane
- Phospholipases and kinases use PH domain and have a direct role in lipid signalling.
How do we study protein protein interactions?
Biochemically
- Centrifugation, chromatography, pulldown reactions
Structurally
- X ray crystallography, NMR, electron microscopy
What is the role of a tag in affinity chromatography or immunoprecipitation?
Tag the protein of interest with a negatively charged flag peptide and have an antibody against the tag
- Can be used for rapid purification of tagged protein and many associated proteins
What is the immuno pull down of interacting proteins?
- Mix tagged protein with cell extract or other proteins to allow binding of complexes
- Add antibody to tag
- Add protein A coated beads
and centrifuge to recover complex. - WASH beads.
- Identify by mass spectrometry
or western immunoblotting. - Use a specific antibody targeted to the epitope tag attached to the protein of interest.
- The antibody protein complex is recovered using Protein A coated beads. Protein A is a bacterial protein that binds strongly to immunoglobulins.
- The Protein A beads are used to ‘immunoprecipitate’ the protein complex
What are the common tags used for affinity chromatography or pull down?
Glutathione-S-transferase (GST)
Hexa-histidine (6xHis)
What are the common tags used for immunoprecipitation?
HA peptide YPYDVPDYA
Myc peptide EQKLISEEDL
Flag peptide DYKDDDDK
What is GST affinity pull down?
- Recombinant DNA techniques are used to make fusion between a protein and glutathione S-trandferase (GST)
- Use fusion protein to ‘pull’ an interacting protein from a mixture, known as ‘pulldown’ in this case GST-pulldown.
- Resulting mixtures separated by SDS-PAGE and western blotted with antibodies for binding partners.
- If binding partners unknown, identify by mass spectrometry
What is yeast two-hybrid screening used for?
To identify interacting sequences
Outline yeast two hybrid screening
- Bait protein cDNA cloned into bait plasmid in the host yeast strain.
- A large library of random DNA prey plasmids are added
- Interaction between bait and prey after introduction into yeast allows DNA binding and activation domains to activate a reporter gene allowing it to survive
- Yeast colony survives on a restrictive growth media
- Correct Prey plasmid recovered and sequenced to find interacting protein.
What is the role of ELISA (enzyme linked immunosorbent assay)?
To measure the strength of protein interactions
Outline direct and indirect ELISA as a method of measuring the strength of protein interactions
Direct ELISA
- Antigen is attached to a 96-well plate
- Primary antibodies added to the plate. These antibodies have been tagged with a fluorophore or an enzyme, allowing the target protein to be identified
Indirect ELISA
- Secondary antibodies used to detect the primary antibodies by binding to the Fc domain
These antibodies have been tagged with a fluorophore or an enzyme
- Allows for amplification of the signal if protein is present in small amounts
What is fluorescence resonance energy transfer (FRET)?
It measures the strength of protein interactions
- The two proteins are labelled with blue fluorescent and green fluorescent protein
- If resonate green light then the proteins must be interacting with each other
- Because the green fluorescent protein absorbs blue light and admits green (have to be close enough to protein labelled with blue to absorb the light)
What is surface plasmon resonance?
Light source is projected onto a sensor chip and is reflected back to a detector
The light source causes surface plasmon resonance (electron resonance) which can form a trace
The binding of prey molecules to bait molecules on the sensor chip increases refractive index of the surface layer and therefore alters the resonance angle for plasmon induction. This shifts the position of the trace
The Kon (association rate) and the Koff (dissociation rate) of a given protein protein interaction can be read directly from these traces
Give the equation for the association rate of protein interactions at equilibrium
Kon[A][B] = Koff[AB]
[AB]/[A][B] = Kon/Koff = K = Equilibrium constant
Give an example of how protein protein interactions can be studied in vitro/
Vesicle movement along microtubules relies on kinesin interaction with microtubules and can be studied by light microscopy
- Can attach the head domain of the kinesin to glass plate to study how the microtubules move
How is DNA usually damaged?
Oxidation, hydrolysis or uncontrolled random
methylation of any of the four nucleotides
What can DNA damage lead to?
Change the bases coding properties, remove a base from the DNA and actually fragment the DNA by breaking the phophodiester backbone
What is the most common form of DNA damage?
Hydrolytic deprurination and deamination of bases
What occurs after the deamination of cytosine?
Become uracil
- Means that the base will be mispaired
- In DNA replication, one parental strand will therefore instruct DNA polymerase to pair it with adenine
What occurs after the depurination of adenine?
Adenine has been removed by hydrolysis
- Sugar and backbone still there but no base
- Meaning one parental trans in DNA replication will have lost adenine so there will be a full base pair deletion in that newly synthesised DNA sequence
How are deaminated and depurinated bases repaired?
Base Excision repair
Explain the base excision repair pathway
Where cytosine is deaminated to uracil, uracil DNA gycosyclase recognises this abnormally as the uracil in the original DNA strand is mispaired with a guanine which is recognised
- Uracil DNA glycosyclase then removes the uracil base
- Acts as a signal to activate two other enzymes
- The AP endonuclease removes the deoxyribose sugar and the phosphodiesterase removes the phosphate creating a single nucleotide gap
- DNA polymerase can then fill in the gap and use the guanine from the complementary DNA strand as a template to fill the gap with cytosine
- DNA ligase then seals the nick
What can ultraviolet radiation cause?
The formation of Pyrimidine Dimers, which can arrest DNA Replication or cause mis-reading of the DNA sequence by DNA Polymerase
Causing the formation of covalent binds between carbons in adjacent pyramiding rings - distorting the DNA and DNA polymerase ability to recognise the nucleotides
How is DNA damage by UV radiation fixed?
Nucleotide excision repair
- Excision nuclease is activated and creates two cleavages in the backbone, on either side of the damage
- DNA helicase then removes this fragment that he excision nuclease has created displacing several undamaged bases along with the damaged pair
- Recognised as a primer template junction (gap) for DNA polymerase and extends across the gap
- DNA ligase then seals the nick
What can defective nucleotide excision repair machinery lead to?
Makes skin vulnerable to UV light
People with these mutations have hypersensitivity to skin cancer
- Xeroderma pigmentosum - can be caused by mutations in the following genes - XPA, XPC, XPD, XPF, XPG
What are the homologues of E.coli Xeroderma pigmentosum proteins?
Uvr proteins:
UvrA, UvrB, UvrC, UvrD
Where does nucleotide excision repair usually occur?
It is tightly coupled to areas being transcribed
- Rapid response
- transcriptively active genes
- Enzymes involved (such as excision nuclease) are associated with RNA polymerase II
Why are double stranded DNA breaks dangerous?
Because it could lead to large fragments of chromosomes being lost
How are double stranded DNA breaks formed?
Ionising radiation
How are double stranded DNA breaks repaired?
- Non-homologous End Joining
2. Homologous Recombination (HR)
What is Non-homologous End Joining?
A rapid way of the body repairing double stranded DNA breaks
- rapidly joins together free ends of fragments that are in close proximity to each other
What are the risks associated with Non-homologous End Joining?
May join the wrong fragments together which can lead to loss of DNA fragments
What is homologous recombination?
The last line of defence in DNA repair
- It makes use of information that is in the undamaged homologous chromosome - high accuracy
Where is homologous recombination commonly needed?
In other forms of repair where there is a partial single stranded section of DNA - vulnerable to stress so may break completely
Outline the mechanism of homologous recombination
- Two sister chromatids (one damaged, one not) accurately align
- Exonuclease is activated and degrades the 5’ ends of the damaged chromatid to create two long 3’ overhangs
- RecA then promotes strand invasion of the undamaged template molecule by one strand from the damaged DNA molecule acting as a primer
- The complementary sequence to the 3’ overhang on the undamaged chromosome is displaced with the damaged one (forms a holiday junction)
- The 3’ end of the invading strand acts as a primer for DNA polymerase
- DNA polymerase extends the 3’ end cross the sequence that has been rendered single stranded on the other fragment - branch point mutation
- When it has all been copied, DNA helicase displaces the extended molecule and promotes its reanealing to the damaged DNA
- DNA polymerase synthesises across the gap on damaged chromatid using the newly synthesised extended molecule as a template
- DNA ligase seals the nick
What types of cancer can mutations in the components in homologous recombination lead to?
- BRCA2: breast, ovarian and prostate cancer
- ATM: Ataxia telangiectasia – leukaemia, lymphoma
- Fanconi Anaemia – complicated with leukaemia: 13 different FANC genes
How has knowledge about homologous recombination and cancer lead to cancer drug development?
Cells that are defective in this way are heavily dependant on other pathways for DNA repair
- Can therefore develop drugs that target other forms of DNA repair
- If these cells cannot use homologous recombination to other forms of DNA repair then those cells will die (synthetic lethality)- killing the cancer cells
What is a holiday junction?
The crossing over of one strand from one helix forming base pairs with the complementary strand of the other helix
How is homologous recombination used in meiotic cells?
Creates new combinations of alleles
- Create a double stranded break in one of the two chromosomes using the endonucleases: Spo11 (makes the initial cleavage) and Are11 (resects the 5’ ends, creating 3’ overhang)
- RecA is recruited and promotes formation of holiday junction (strand invasion)
- DNA polymerase fills I the gaps on both DNA strands using the DNA from the other chromatid as a template
- DNA ligase creates the second holiday junction
- The holiday junctions are then resolved and crossing over is complete
What is the main difference between homologous recombination in repair and in meiosis?
DNA polymerase uses strands from both chromatids as templates to fill in self created breaks in meiosis
What are the two possible ways that holiday junctions can be resolved in meiosis and which is used?
Pathway 1
- Internal strands of holiday junctions are broken and rejoined (same strands are broken and rejoined at each junction)
- This doesn’t achieve crossover as all four stands need to be broken - not used
Pathway 2
- At the holiday junction, the two outer strands are broke and rejoined but at the other holiday junction, the internal strands are broken
- The chromatids then rejoin with a successful crossover as each chromatid has a part of the other chromatid on it
How often does cell proliferation occur?
Typically one division every 24hrs in proliferating mammalian cells. Other cells do not divide once they are born and last for many years.
What are the four stages of the cell cycle?
G1 - Gap 1
S - DNA replication
G2 - Gap 2
M.- Nucleair division
What stages of the cell cycle make up interphase?
G1, S, G2
What occurs during M phase of the cell cycle?
Prophase – condensation of sister chromatids (identical copies).
Metaphase – attachment of the mitotic spindle to the kinetochore by microtubules.
Anaphase – separation of sister chromatids.
Telophase - Nuclear membrane reforms
What are the types of cell division in yeast?
Fission and budding
What are the advantages of using yeast as a genetic model for cell cycle?
Rapid division rate <1hr
Cell cycle control genes are highly conserved
Yeast can be grown as haploids or diploids
What genetic tricks can we use to allow for identification and investigation of potentially lethal mutations?
- Diploids can be used to maintain lethal mutations (they won’t die as there is a second wild type gene) and then they can be studied as haploids
- Temperature sensitive mutations allow growth at a permissive temperature - vary the temperature depending at what stage in the cell cycle you wish to investigate
What are Cdc genes?
Cell division cycle genes
What are the advantages of using Xenopus leaves as a biochemical model for cell cycle?
Easy to collect eggs
Rapid division rate
Large size makes purification of proteins easier
Can be manipulated by injection of RNAs or chemical into the oocyte
What is meant by cell free mitosis and what are its uses?
Cytoplasm from frogs egg is added to the nuclei from a frogs sperm and ATP is added
Mitosis
One can deplete the cytoplasm of different proteins using antibodies
One can remove cytoplasm at different stages to study changes
(eg in protein phosphorylation) over time.
When are the checkpoints in the cell cycle?
Start checkpoint
- End of G1
- Is the environment favourable for cell division?
G2/M checkpoint
- As enters mitosis
- Is all the DNA replicated?
- Is the environment favourable for cell division?
Metaphase to anaphase transition
- trigger anaphase and cytokinesis
- Are all chromosomes attached to the spindle?
What are cyclins?
- Cyclins are proteins that are expressed at different levels during the cell cycle
- When present, cyclins bind to specific kinases (called cyclin dependent kinases, Cdks) to activate them.
- CDKs phosphorylate many proteins that are specific to certain stages of the cell cycle.
What other proteins also modify CdK activity?
Wee1 kinase phosphorylates Cdks to inactive them
Cdc25 is a phosphatase that removes the phosphate group and activates Cdks
What are APC’s (anaphase-promoting complex)?
A ubiquitin ligase
It ubiquinates M-cyclin, S-cyclin and securin
Involves E1 and E2 enzymes which cause ubiquitination and therefore degradation of the cyclin in the proteasome
What is a securin?
A protein that holds together sister chromatids
What is meiosis?
- Diploid organisms have two versions of each chromosome (homologues).
- Homologues are either paternal or maternal.
- Only one homologue for each chromosome is packaged into a gamete.
- Meiosis resembles mitosis except that there are extra steps that segregate homologous chromosomes.
- Pairing of homologues before segregation allows for crossing-over (homologous recombination)
What occurs in meiosis I?
Crossing over and segregation
- DNA replication
- Homologue pairs line up on spindle and are pulled apart
What occurs in meiosis II?
Resembles mitosis
Main difference to mitosis is that cells in meiosis II are haploid not diploid
What occurs in meiotic prophase I?
Homologues pair up
- Pairing in facilitated by the synaptonemal complex (proteins) as well as DNA base pairing between homologues.
- Homologous recombination between nonsister chromatids serves two purposes:
1) It aligns the chromosomes up ready for anaphase and facilitates formation of the synaptonemal complex.
2) It allows for genetic recombination between paternal and maternal DNA on the same chromosome
Why is genetic variation important?
For evolution
What are the problems associated with meiosis 1?
Mistakes during meiosis I result in gametes with an extra chromosome or lacking a homologue.
This is called nondisjunction and the cells that arise from these gametes are called aneuploid.
4% of mammalian sperm is aneuploid, 20% of mammalian eggs are aneuploid.
What is the normal relationship between the level of transcription and level of expression?
Usually increased transcription means increased expression
What can occur that stops increased transcription meaning increased expression?
RNA may be transcribed but not translated into a protein
Protein may be readily degraded
What kind of interactions are essential in the regulation of transcription?
DNA protein interactions are essential for regulation of transcription
How do DNA binding proteins interact with DNA?
DNA binding proteins are positively charged which allows the protein to stick to the negatively charged backbone
- The charge allows the protein to be in close proximity to the DNA molecule but the alpha helicies reach into the major groove to interact with specific bases
What amino acids are responsible for DNA binding proteins having a positive charge?
Lysine or Arginine
