1
Q
What were the experiment that lead to the conclusion of DNA being the genetic material?
A
1928 - Fred Griffith found that material from heat killed virulent bacteria could transofrm non-virulent
144 Oswald Avery
Frationated the isolated material to demonstrate that they were nucleic acid and that they didn’t transform when digested
1952 Alfred Hershey
Radioactively labelled with P and S in bacteriophages and found that the P was used in phage progeny and S was in the ‘ghosts’
2
Q
What is Base tautomerisation?
A
Bases can exist ad tautomers where proton in the base has migrated to a different place and they have implications for DNA replication accuracy and provide variation.
99.9% common form
0.01% tautomers
3
Q
What is a nucleoside?
A
A nucleoside is the base-sugar comlex and it becomes a nucleotide when attatched to phosphate groups.
4
Q
What were the clues that DNA in cells is in a double helix structure.
A
Chargaff did studies on base composition and found that the percentages of A and T and G and C were usually the same.
And that ammounts of purine bases = ammounts of pyramidine
Franklin and Wilkins then did X-ray diffraction analysis which showed interwtined helicies and suggested dimensions from the spacing of the lines (3.4nm per base turn and a diameter of 2nm)
Watson and Crick then used model building to propose the right-handed helix antiparallel structure infering the bonds between base pairs.
The structure of B-DNA was confirmed in 1980 using oligonucleotide duplex and confirmed diameter is 2nm but that there were 10.5 base pairs in a turn but still 0.34nm between pairs
B-DNA forms major and minor grooves
Most DNA is B-DNA but A-DNA has 11bp/turn and has even sided grooves and Z-DNA which is a left handed helix of 12bp/turn
5
Q
What is supercoiling in DNA?
A
Open uncoiled DNA is relaxed and Supercoiled DNA is under tension
Supercoils can be introduced when a circular molecule is cut and held while one end is twisted.
When reatatched the DNA twists to restore number of base pairs per turn wrapping around itself
This can be quatified using the linking number (Lk) and is a product of Twist (TW) which is the number of turns and Write (Wr) which is the number of supercoils and can be negative
6
Q
Define genome size
A
The genome size is the total amount of DNA contained within one copy of a single genome (e.g haploid count in diploid organisms)
7
Q
How varied are viral genomes?
A
Very
Can be DNA/RNA
Single/double stranded
Circular/linear
may overlap
8
Q
What are the ranges of the abundence of DNA sequences?
A
Unique (one to a few copies)
Moderately repetitive (few to 10^5)
Highly repetitive (10^5 to 10^7) copies per genome
9
Q
How is DNA organised in bacterial chromosomes?
A
DNA in bacteria is much longer than the cell so it must be organised and compacted.
It has no introns
Small positive proteins binds along DNA to counteract negative charges allowing for more supercoiling.
10
Q
Describe Eukaryotic DNA packaging
A
DNA is wrapped around 4 core histones which are rich in postiviely charged lysine and arginine to counteract negative DNA
The DNA is wrapped around a histone complex to form a nucleosome
The histone complex is the histone octamer which has two of each 4 core histones
146bp of DNA wraps around the octamer and supercoils around 1.75 times
Removing histone complex leaves negatively supercoiled DN which allows for easier seperation.
Each histone core has a N-terminal tail (25 AA in length) that extends outwards etween DNA coils and these interact with other nucleosomes and can be modified
Histones can form higher order structures (10nm and 30nm fibres) which is a regular arrangment that compacts them.
Histone H1 is involved in formation of the 30nm
but the 30nm fibres might not be that important
Histones can be modified which can affect chromatin structure and function and this modification is reversible
11
Q
Describe chromosome structure and compaction
A
It undergoes various phases of compaction
decondensed regions stain lightly (euchromatin)
Chromatin in more compacted regions stain more darkly - heterochromatin
Chromatin structure affects:
Transciption (from euchro to heterochro)
Replication
Recombination
Chromosome trnasmission
Chgnges in chromatin are key to gene activity regulation (eucho is more acetylated than heterochro)
12
Q
Explain the difference between the lagging strand and the leading strand in DNA replication
A
DNA is replicated from a 5’ to a 3’ direction and the leading strand is seperated in the opposite direction of this allowing for continuous replication
The lagging strand is split from 5’ to 3’ so the opposite direction synthesis strand happens in the opposite direction and will reach the end of the DNA so a new one will replace it as new DNA is opened up.
Both sides of replication have a lagging and leading strand as it starts in the middle and travels in both directions on both sides from the starting point.
13
Q
What are the three phases of DNA replication?
A
Initiation is the first phase where the origin of replication is recognised by initator proteins that open the helix and recruit helicases. Helicases unwind the dna.
Primers are synthesised in this phase to kickstart synthesis.
Elongation of replication is the second phase. The sliding clamp is recuited to the 3’ end and polyemerase associated with this clamp.
Complementary bases are added to the strand
Termination of replication is the final stage and occurs when polymerase encounters replicated DNA, where two replication forks meet or when it reaches the end of the chromosome. At witch the replication complexes are dissasembled
Primers are removed and replaced with DNA and DNA ligase connects adjacent strands.
14
Q
Decribe the right hand motif of the DNA polymerase
A
The DNA polymerase has three domains described as thumb, fingers and palm
The palm contains the catalytic sidte for nuclotide addition.
The fingers helps position the incoming nucleotide
The thumb holds elongating dsDNA maintaining contact.
15
Q
What are the main DNA polymerases?
A
DNA polymerase III in bacteria for leading and lagging
Eukaryotes:
DNA polymerase δ (lagging)
DNA polymerase ε (leading)
16
Q
What is the mechanism of DNA polymerase?
A
It catalyses a phosphoryl transfer reaction
attacks 3’ OH on α-phosphate of dNTP and the hydrolysis of the released pyrophosphate provides the energy for the reaction.
17
Q
Descibe DNA polymerase active site specificity and proofreading
A
Specific complementary base pairs are the only bases to correctly fit and mismatches have different shapes that don’t fit
These active sites are highly conserved and are therefore grouped according to evolutionary lineage of the rest of the protein.
error rate of <1 in 100,000
for these errors proofreading exonuclease activity removes mismatched bases from the 3’ end of DNA.
This is done as the exonuclease has increased affinity for 3’OH only when the incorrect nucleotide is present. This requires energy to remove and replace.
18
Q
Describe DNA helicases and the differences between bacterial and eukaryotic helicases.
A
DNA helicases catalyse the unwinding of the DNA helix travelling along it at the fork.
They are hexameric ring proteins that move along ssDNA
Bacterial helicase travels 5’ to 3’ and Eukaryotic travels 3’ to 5’ suggesting independent evolution
Bacterial DnaB helicase is loaded onto ssDNA by DnaC helicase loader complex and get right to work
Eukayotes loads MCM2-7 complex helicase in G1 for maintenance and CMG DNA helicase is assembled and activated in S phase.
19
Q
How does DNA stay uncoiled and prevent secondary structures forming fom seperated single strand DNA?
A
DNA-bnding proteins bind to single stranded DNA after helicase seperates it to keep these strans open and protect them from nucleases.
Topoisomerases release overwound DNA by transiently breaking DNA allowing supercoils to relax.
Important as helicases introduce tortional stress as they approach supercoils.
Bacterial DNA gyrase (type II) introduces negative supercoils
20
Q
What are the 3 types of topoisomerases and what do they do?
A
IA cut one strand and bond to 5’ end of the DNA until the ends are rejoined
IB ut one strand and bind to 3’ end of DNA until the ends are rejoined and it is released
Type II cut both strands and join to 5’ ends od DNA requiring ATP and is relesed upon rejoin
Bacterial Type II (DNA GYRASE) maintains negative supercoiling.
Topoisomerases are also required for unlinking replicated circular chromosmes.
21
Q
What is the origins or replication and how are they relevant to other systems in replicaiton?
A
They are the point where dsDNA is seperated and replication is initiated. Bacteria have one per chromosome and eukaryotes have multiple.
Initiator proteins bind to the origins and recruit helicases.
In bacteria DnaA binding at oriC recruits DnaB helicase to unwind DNA
In most eukaryotes origins are not defined by specific sequences and can be initiated and multiple sites with binding sites being influenced by multiple factors
Bacterial primase (DnaG) synthesises a primer which is added by DNA polym.. III
The origin unwinding occurs in two stages. They are licensed in late M / early G1 where the origin is established and when MCM2-7 is loaded is it considered licesed and only happens once per cell.
After this DNA helicases are loaded at the origins in G1 and activated in S-phase. Once s-phase begins the MCM2-7 complex is phoshorylated allowing recruitment of Cc4 and Sld3 (see slide 8 lecture 4)
DNA replication doesn’t start until the origin is unwound and helicases are activated.
There is one primer required at originson the leading strand but several primers on the lagging strand.
Initiator DNA is added by the aplha subunit of polymerase
22
Q
What is the sliding clamp and what is its function in DNA replication?
A
DNA polymerases for the entire chromosome stay attatched for thousands of bases
Association of a sliding clamp processivity factor increases the rate
The clamp binds to DNApolymerase and keeps it tethered to the DNA while sliding along with it.
In eukayrotes the clamp is PCNA and interacts with DNA polymerases δ and ε
In prokaryotes the clamp is β protein and interacts with bacterial DNA pol III
But the structure of these clamps areconserved
They are loaded onto the 3’ end of RNA or iDNA primer by clamp loaders.
γ-complex in bacteria
Replication Factor C (RFC) in eukaryotes
some of the clamp loaders are AAA+ ATPases as ATP is required for the clamp loader to associate with the clamp in a way to open the clamp.
The loading of clamps facilitates polymerase switiching when it binds to the primer from Pol α–primase to DNA Pol δ or DNA pol ε
23
Q
How is the leading and lagging strand in DNA replication coordinated?
A
In bacteria the replisomes flexible tau subunits ensures both strans DNA Pol III are associated at the fork even when released after each okazaki fragment.
In eykaryotes they are not directly linked but multisubunit Ctf4 acts as a hub to couple CMG helicase, and both polymerases at the fork
24
Q
How are okazaki fragments joined after synthesis?
A
In bacteria DNA pol III disassociates when it reaches the next primer and the sliding clamp remains to associated with DNA pol I to reomve the primer and leave a nick for DNA ligase to seal
In eukarotes DNA pol δ keep synthesising when it meets the RNA primer displacing it leaving a flap cleaved by flap endonuclase (fen1) and DNA ligase I seals the nick
25
How does termination of replication occur in bacterial chromosomes?
Termiation occurs at Ter sites each Ter site is bound by a single Tus molecule and they STALL the fork. One sides replications stops at the complex while the other fork continues until they converge. DNA polymerase I and DNA ligase complete replication after termination
26
How does termination of DNA replication occur in eukaryotes?
It occurs at multiple sites where replication forks converge CMG complexes move past each other and DNA polymerase δ, Fen1 and DNA ligase I are recruited to complete maturation.
Entwined DNA is respolved by topoisomerase II
27
What are the function of telomeres?
They protect the ends of linear chromosomes.
They have simple repeating sequences and range in length from 50 to >20000.
They exist to form a t-loop to prevent DNA replication mechanisms from trying to repair the end of the DNA as if it was a DNA break.
Without telomeres polymerases will attempt to bind the ends of chromosomes together creating long chains of chromosomes.
Telomerase is active in tissue-specific stem cells and germline cells and is reactivated in many cancers
28
What is the end-repliation problem?
The DNA replication machinery cannot fully replicate the ends of chromosomes leading to gradual DNA shortening every division.
Telomerease mitigates ths problem by synthesising one strand of the DNA (g-rich 5' to 3' strand)
This elongates the 3' end of telomeres leaving enough space for polymerase to synthesise the portion of DNA it couldn't before.
Telomerase is active in tissue-specific stem cells and germline cells and is reactivated in many cancers
29
How is replication regulated in E. coli?
Initiation of E. coli is co-ordinated with growth rate.
It takes ~50 mins to replicate the bacterial genome but E. coli can divide every 20 minutes. Meaning that a second round of replication starts before the first round is completed.
Re-replication is regulated and intiation is synchronised between both replicaitons.
This is done via DnaA binding at oriC and unwinding DNA when bound to ATP.
After initiation sliding clamp stimulated hydrolysis of DnaA-ATP to ADP
DnaA-ATP then dissociated and can't reinitiate.
Regulatory innactivation of DnaA blocks replication initiation.
11 GATC sites in oriC overlap DnaA boxes and go from fully methylated to hemi-methylated after replication
SeqA binds to hemi-methylated GATC sites and essentially delays DnaA rebinding and prevents more methylation for about 10 minutes
DnaA can bind to datA and datA squesters about 25% of free DnaA
After replication datA is duplicated sequestering 50% making it unavaliable to bind to oriC
DARS recycles DnaA-ADP to DnaA-ATP
30
How is replication regulated in Eukaryotes
During S-phase DNA must replicate exactly once, Incomplete replication, chromosome segregation and re-replication are all bad
Eukaryotic DNA is replicated from multiple origins and origin firing requires MCM loading to establish pre-RC.
MCM cannot load in S-phase
ORIGIN TIMING replates to when an origin fires early or late
ORIGIN EFFICIENCY is the probibility that an origin will fire.
origin firing is mostly random
Intiation of replication is controlled by origin licensing in G1-phase and once the MCM loading proteins are finished they undergo proteolysis to prevent licensing and activation of DNA helicase during S-phase
Different eukaryotes have different prevention methods to prevent MCM2-7 loading outside of G1 (yeast exportes new proteins, metazoa does proteolysis, geminin provides an additional control laer to prevent binding)
31
How are histones recycled to newly replicated DNA?
Histone modifications must be maintained to the daugher DNA for epigenetic inheritance
Parental histones are passed on to aughter DNA and H3 and H4 histone subunits are distributed equally to both daughter cells whereas H2A and H2B are disassembled and reassembled on daughters as dimers.
32
How are nuclosomes formed and modified?
Nuclosome disassembly and reassembly is mediated by histone chaperones that interact with the replication fork proteins.
Each daughter DNA has half the required nucleosomes so new histomes are syntehsised in S-phase
parental histones recruit histone modifaction enzymes to modify new histomes
33
What is DNA damage?
Defined as a change to the regular chemical structure of DNA
I.e:
A break in phosphodiester bacbone
Loss of a base
Alteration to a base
Mismatched base pairs
It can be detected and repaired but often leads to mutation if replicated
One exmple of damage causing a mutation is replication of rare tautomers with mismatched pairs. and slippage via deletion or additon of base pairs
Endrogenous damage:
Losses of purines (deuprination) causes the loss of 18000 bases per cell per day and is more common than depyramidination. These cause random substitutions or deletions which lead to mutations
Deamination can also take place which when done to cytosine ressults in uracil which is not usually present in DNA and deamination of 5 MeC produces a GT pair which becomes a mutation after replication.
Alkylation is where alkyl groups can be added to bases by alkyl donors. Base modifcation can affect base pairing properties causing damage and mutation
Oxidative DNA damage is resposible for high mutant rate in mitochondrial genomes. Bases damaged by reactive oxygen species can be mutagenic or lead to strand breakage and block replication.
34
What are mutations?
Defined as a permanent heritable change in the genome
Can be point mutations or chromosome mutations
Point mutations:
Transition mutations:
Purine to purine / pyramid to pyramid
Transversion:
Purine to pyrimidine and vice versa
Types are:
Silent (same AA made)
Missense (change of AA can be conservtive if similar or non if not)
Nonsense where it codes a stop codon in the wrong place.
Forward mutations are from wild type to defective mutant
Reverse mutations are when a defective mutant turns to a wild type (true reversions is to the same amino acid partial is a similar one)
Supressor mutation is a different mutation that compensates for an inital one
Most mutations are spontaneous (1 in a billion for prokayrotes and 1 in 100 million in germline cells in eukaryotes. (somatic rate is higher and varies)
Mutations are random, not adaptive
Spontaneous mutations occur when damaged DNA is replicated or by replication errors but there are multiple mechanisms to ensure fidelity of DNA synthesis and prevent this. One exmple of damage causing a mutation is replication of rare tautomers with mismatched pairs. and slippage via deletion or additon of base pairs
35
What are mutagens?
Mutations can be induced by exposure to mutagens which cause DNA damage.
Chemical mutagens:
Base analogs
Base modifying agents
Intercalating agens
Physical mutagens:
Ionising radiation
UV radiation
Base analogues can iduce transition mutations by changing existing bases to rare states and making mismatches.
Base modifying agents can damage the DNA such as nitrous oxide deaminating DNA (specifically A C and G). Or Hydroxylamine which hydroxylates and Methylmethane sulfonate which metylates guanine.
Base modifuing agents might need to be activated metabolically before they can damage DNA such as polycyclic aromatic hydrocarbons metabolised to epoxide compounds. These cann be found in meat and most smoke.
Intercalating agents induce frame shift mutations by extending and partially unwinding the DNA helix causing insertions and deletions on replication.
36
How does radiation act as a physical mutagen?
UV RADIATION:
UV is split in UVA B and C
UVC is the most mutagenic but is all absorbed by atmospheric oxygen.
UBA and UVB ar the most biologically important as 10% of UVB reaches the surface and most UVA reaches the surface.
DNA absorbance of UV radiation peaks at 254 nm which is in the UVC range.
UV radiation only affects the pyrimidines and not the purines. It causes intrastrand cross-linked pyrimidine dimers which physically constrains and distorts DNA affecting polymerase function and introducing mismatches.
IONISING RADIATION:
Short wavelength high energy radiation from natural, pheraputic, diagnostic or ocupational sources.
35% od DNA damage is from direct interaction of radiation energy with DNA and 65% occurs indirectly via reactive oxygen species fored by ionisation of water.
Ionising radiation:
damages bases,
Breaks polynucleotide strand phosphodiester backbone and has lethal effects due to double strand breaks as there is a loss of continuity.
There are >100 different types of base damage from ionising radiation identified.
37
What is the Ames test?
Ames test is a test for mutagenicy.
It is done by adding salmonella auxotrophic for histidine (doesn't produce it) so no colonies can be formed in a medium without histidine.
The suspect mutagen is added (usually with liver extract) and added to medium.
Increased colonies are produced in mutagenic presnece due to mutations producing his. (some produced via spontaneous revertant mutation but increased due to mutagenicy)
38
How do cells repair DNA damage?
Direct reversal of damage:
1) Repair of alkyation damage. Alkylation on guanine or DNA backbone is repaired by alkyltransferases.
(The E. coli alkyltransferase is Ada aka O6-Methylguanine-DNA Methyltransferase)
2) Enzymatic photoreactivation of pyrimidine dimers by photolyase enzyme.
This is found in all organisms besides placental mammals.
It uses blue light and chromophores to split pyramidine dimers (produced by UV rad) via electron transfer using FADH
Excision of DNA damage:
1) Mismatch repair. Done when it recognises mismatched base pairs and are able to discriminate between the correct (parental strand) and the incorrect base in a mismatched pair.
It then excises the base and carries out repair synthesis.
This is done by MutS recruited to the mismatch and MutH recognizes the new DNA by which one isn't methylated. The MutL interacts with MutH and forms MutSLH which causes MutH to create a nick in the new strand which is unwound by helicase, digested by exonuclase and repaired by DNA pol III
This process can also repair insertions and deletions.
2) Base excision repair. Cellular glycosylases are specific to a particular type of damage (we have 11 so only the most sigificant damages) and these are highly conserved. The glycosylase cleaves the bond from the sugar creating an abasic site.
AP endonuclases cleave the backbone and the gap is then filled by polymerase and ligated.
3) Nucleotide excision repair. Specifically for helix distortion (Not other damage)
Bulky lesions that distort the helix repaired this way. In prokaryotes UvrA and UvrB scan for distorted regions and UvrB unwinds damaged regions. UvrC then nicks the damaged DNA and damaged DNA is removed by UvrD where DNA can be resynthesised form the undamaged strand. Similar in eukaryotes and all organisms have this function but with different proteins. It is very accurate and does not induce mutations.
The regions of DNA that are being actively transcribes are repaired preferantially. Inherited defects can cause a rare fatal disorder that gives predisposition to skin cancer.
39
What happens if DNA isn't repaired before replication?
Types of damage:
Mismatch base pair damage results in mutation via normal replication machinery.
Altered/absent bases - DNA lesions - DNA damage tolerance
Some lesions can be replicated via normal machinery but will miscode the DNA.
Other damaged bases can't be replicated and lead to fork stalling (unable to proceed past the legions).Fork stalling induces the DNA damage response recruiting "Specialised translesion synthesis DNA polymerases" will replicate some DNA with damaged template.
TLS DNA polymerase have more open and flexible active sites allowing for low fidelity synthesis of damaged DNA. (much higher error rates)
TLS DNA polymerases are recruited to forks by interaction with sliding clamps
The DNA damage repsonse increases DNA repair protiens
Delays cell cycle and can trigger programmed cell death in multicellular organisms
40
How does the DNA damage response work in bacteria?
Bacteria respond to DNA damage with the SOS response to induce >40 proteins.
RecA and LexA are central
RecA is a multifunctional DNA binding protein that acts as a damage sensor
RecA is inactive in the absence of damage
LexA is a repressor that prevents too many SOS genes from being transcribed
In fork stalls RecA binds to form a filament and becomes activated cleaving LexA. This prevents LexA from repressing and transcribes SOS genes which encode DNA repair proteins and inhibits cell division increasing the time before division.
After the DNA is repaired DinI helps reestablish supression by mimicing DNA and RecA binds to it and is sequestered and therefore LexA is free to bind to SOS geness and repress them
41
How does DNA recognize DNA damage
DNA damage sensors recognize damage DNA and recruit specific tranducer regulaatory kinases to the site which activate downstream proteins that recruit effector proteins to repair damage and halt cell cycle
e.g RPA is a damage sensor and binds to single stranded DNA and the fork and is removed as the lagging strand is replicated. DNA damage cause polymerase to fork stall and RPA remains bound
RPA rectuits ATR and ATRIP binds to both. RPA also recruits a repair specific sliding clamp-clamp loader complex (9-1-1 complex) this activates ATR via TOPBP1
Activated ATR phosphorylates targets to modulate DNA metabolism
RPA also recruits a complex that monoubiquitinates PCNA as polys have reduced affinity for ubiq PCNA and TLS polys have affinity for them resuming damaged DNA synthesis.
double strand breaks are sensed by the MRN complex which interacts at the break and can hold the ends together. This recruits the regulatory kinase ATM which interacts with the MRN/DSB complex and autophosphoyrlates and activates.
Activated ATM amplifies the DSB and phosphorylates targets to modulate DNA metabolism and the cell cycle.
Double strand breaks are also sense by the KU heterodimer which is abundant and binds to the DNA ends strongly and recruits the regulatory kinase DNA-PKcs forming DNA-PK which recruits proteins to rejoin DNA via non-homologous end joining (NHEJ) which causes some loss of information at the DSB site.
ATR, ATM and DNA PKcs are all structurally related.
42
How does DNA repair double-strand breaks?
Two alternate pathways:
Non-homologous end joining (NHEJ) which simpl rejoin the end (predominant in non-dividing cells). It is the only readily avaliable G1 path and removes a few nucleotides before ligation. Therefore information is lost and NHEJ is mutagenic and the non-prefered method.
Homology-directed repair uses homologous DNA as a template usually in late S phase/G2 when a sister chromatid is avaliable as a template.
43
What is the difference between Homology-directed DSB repair and non-homologous end joining?
Homology-directed DSB repair uses existing DNA as a template and can't occur in G1. This is the preferred method and involves pairing of DNA duplexes accurately and is well conserved.
Four phases:
1) Generation of ssDNA at the DSB (presynsapsis)
2) Pairing by one of the single-stranded ends invading an intact homologous dulex (synapsis)
3) Reair of the damaged duplex by DNA syntehsis (postsynapsis)
4) Seperation of the two suplexes (postsynapsis)
put simply single stranded DNA is generated at the break via helicase and exonucleases. These then form a D-loop in existing DNA and DNA is synthesised based on the sister chromatid to create DNA that can bind with the other break and nicks are ligated to complete the repair.
Non-homologous end joining has no undamaged strand to act as a temlate and simply rejoins the ends resulting in loss of information.
44
What are the key proteins in mediating homology directed DSB repair (in E.coli)?
Long single stranded tails are generated by helicases and nucleases (presynapsis). the RecBCD complex in E.coli has both these functions and attatches at the DSB and moves along the DNA unwinding and degrading it.
RecBCD is modulated by Chi sequences in the DNA
Chi is an over-represented 8bp sequences that occures around once every 5kbp.
It causes RecBCD nuclease activity to decrease and for the 5' strand to be preferentially degraded creating a 3' end that RecA binds to.
RecA is E.colis verion of eukaryotic Rad51
RecA loads onto the single-strand forming the presynaptic filament (SOS response).
The helical nucleoprotein filament distorts the DNA extending it by up to 50%
In vitro RecA does strand exchange but in vivo more proteins are involved to prevent secondary DNA to impede reactions.
BRCA1 and BRCA2 are accessory proteins to thi sprocess and defects in these are linked to breast cancer
This is a high fidelity repair mechanism.
45
How can homology directed repair result in gene conversion?
When sister chromatids aren't avaliable (as in G1) homogous chromosomes are used but they slightly differ in sequence and the repair now has the sequence of the homolog not the original (gene converion)
46
What is homologous recombination?
It uses the samc emachinery that does homology-directed repair to generate new deliberate combinations of genetic info. Can occur between any DNA with extensive regions of identical/similar sequences
This is used in meiosis 1 to generate diversity and producing unique gametes. and allows gene transfer in bacteria via conjugation and transduction.
It is initiated by an intentional DSB and then the 3' tail invades the homologous duplex (i.e 2 d loops at once) and new DNA synthesis occurs.
Two intact dsDNA regions joined by two Holliday Junctions are formaed.
Holliday junctions are four-armed cross-stranded junctions (see lecture recording). They can be moved along the recombining DNA by branch migration
(in bacteria branch migration is driven by RuvAB complex)
RuvA opens the junction to the square planar configuration
RuvB is a hexameric helicase which moves the holliday junction along the DNA.
Holliday junctions are resolved by RuvC which cleaves the junction seperating participating DNA either horizontally or vertically. (horizontally results in a non-recombinant product and vertical is recombinant.
47
What are sex plasmids?
(Relatively large ~100kbp) Plasmids such as F (fertility) plasmid of Ecoli in which about 35% fo the sequence encodes functions to permit transfer between bacteria.
The rest contiains 4 insertion sequence elements that mediate the transfer of bacterial genes by conjugation.
Can exist as a plasmid or be integrated into chromosome.
48
What are R plasmids?
Relatively large (30-100kbp) Plasmids that encode resistances to antimicrobial drugs, heavy metals and toxins. (see 243 for more)
49
What are col plasmids
Small plasmids (mostly <25kbp)
Encode biological factors and only transferred if F or R plasmids are present in the same cell.
Useful for molecular biologists as vectors.
50
Explain bacterial conjugation
Conjugation is unidriectional DNA transfer from an F plasmid postive cell to a F- cell with no F plasmid.
Done via a long tubular F-pilus which allows a cytoplasmic bridge to transfer genetic material (encoded by F plasmid)
tra genes in the F plasmid encode contact and DNA transfer functions.
Transfer is initiated by introduing a nick in DNA at the OriT (origin of transfer) and the 5' end is transferred. DNA synthesis then reforms the plasmid in the original cell and the recipient.
If all of the F plasmid is transferred the new cell becomes F+ as well.
Only F plasmid is transferred in F+ x F- interactions.
To transfer bacterial chromosome the F factor must integrate into the chromosome via recombination creating Hfr strains. (these are reversible)
In the reverse the F plasmid is exised from the chromosome. But imprecise excision can generate the plasmid carrying bacterial genes. F plasmid containing an adjactent Lac+ region can be generated and its transfer to recipient cann be maintainted without integration. This is used in genetic analysis of gene regulation (sexduction)
Hfr transfer can then take place starting with the nicking of oriT on the integrated F plasmid and followed by the bacterial chromosome into the recipient creating a new recombinant. (kinda like a big plasmid)
But the bridge is fragile and often breaks so very rare for more than a fraction of the chromosme being transferred.
Non-species specific but dependent on DNA homology for sucessfull recombination.
Conjugation can be used to map bacterial genes via allowing conjugation and breaking it off at various times before testing on selective media to see which genes were transferred.
-interrupted mating experiment
This was used to show that the E.coli map genetic map is circular. via multiple strains integrated at different sites.
51
52
What is the difference between a true pathogen and opportunistic pathogen?
True pathogens can cause infection in individuals with normal host defences
Opportunistic cause infection in individuals with abnormal host defences.
53
How is it determined that a pathogen causes a specific disease?
The pathogen must be present in every case of the disease
The pathogen must be isolated from the host and grown in pure culture
The specific disease should be reproduced when a pure culture of the pathogen is inoculated into a healthy susceptible host
The athogen must be recoverable from the experimentally infected host.
54
List virulence factors and explain how you can exploit them:
Toxins can be used to develop antitoxins and vaccines
Capsules can be used to produce vaccines (such as meningitis)
Adhesins (establishes biofilm such as in UTIs but no exploitation yet)
55
How can changes in the normal flora affect you and how can it change?
Changes in the normal flora can make you more susceptible for infection.
Changes in hormonal phusiology and development can make people (particularly females) susceptible
Taking antibiotics can disrupt the normal flora.
56
How can normal flora from the gut affect you harmfully?
Excape of the normal flora to abnormal site (i.e perforated appendix, gall bladder swelling and UTIs)
Antibiotic use can cause overgrowth of resistant flora including C.Diff (treated by stopping antibiotics or temporarlily swapping to a different type before stopping for recovery)
57
How are can you get opportunistic infections?
Immunosupression (shift from infections fue to gram + to gram - pathogens, increased fungi importance, increased incidence of resistance pathogens)
Breaching defences (Iv, catheter or wound)
Foreign body such as a splinter or prosthesis
Debility such as malnutrition, ill health and old age
58
What is clostridioides difficile (C. diff) and why is it a problem?
It is a gram-positive, rod-shaped, spore forming, anaerobic bacteria that causes symptoms of:
Diarrhoea,
Fever,
Loss of apetite,
Feeling sick,
Pain
People are more vunerable when:
Taking broad spectrum antibiotics,
Staying in a healthcare settings,
Over 65,
Have underlying conditions,
Have weakened immune system,
Have had digestive system surgery.
Its virulence factors are:
Resistance to lysozyme
Adhesisns-
cwp family of proteins
Fironectin-binding proteins
collagen binding proteins
Lipoprotein
Heat shock protein
Invasins-
Proteases
flagellae
30 hours after infecting colon cells damage has started and inflammation and fluid buildup take place.
36 hours after exposure inflamed cells urst and die and spores leave the colon.
59
What are examples of latent pathogens?
Viral:
CMV, Epstein-Barr, Herpes simplex, VZV
Bacterial:
Syphilis, Tuberculosis
Fungal:
Cryptococcus neoformans, Blastomyces dermatitidis
Parasitic: Pneumocysis carinii, Toxoplams gondii
60
How does the spleen affect opportunistic infection?
It is a main antibody production site, filters blood and clears bacteria
After splenectomy sepsis a sever infection can happen withing hours with organ failiure and high mortality
Most common pathogen post-splenectomy is S. pneumoniae
Requires informed patient and staff with alert cards to prevent sepsis
Immunisation to multiple bacteria requicred for prevention. and phrohpylaxis
61
How are infections prevented?
Immunization
Infection control
Screening for latent infection
Prophylaxis
Engineering controls:
(aspergillus prevention)
Filtered air,
Barrier protecion
Protecitve isolaton (HEPA filters)
Respiratory protection
(legionella prevention)
prohibit showers
Implement surveillance
Monitor water supply
initiate decontamination.
62
63
What types of medium are there for bacterial growth (section c)
Minimal medium - only contains defined infredients (bacteria need fully functional biosynthetic pathways)
Complete medium - complex mix of amino acids, sugars, vitamins and essential metabolites (grows all bacteria)
64
How does gene nomenclature work?
Each gene is assigned a lowercase three-letter designation usually abbreviatin gthe pathway/phenotype.
E.g: lac gene affects lactose metabolism
If there is a mutation another letter is added on the end (e.g lacZ)
proteins usually use the gene name but with a capital letter (in bacteria) and is not italicised
See workshop week15 pp for more
65
66
What is specialised transudction?
Transduction mediated by temperate by temperate bacteriophage lambda (phages that can have 2 different lifecycles)
in both the linear phage DNA is circularised and replicated via rolling-circle mechanism producing a concatamer of linear DNA
The lytic destroys the cell as normal
or lyosgenic where the host survives and retains DNA that is integrated into the host chromosome termed a prophage where it replicates as normal until the prophage exits the chromosome and initiates the lytic cycle)
When DNA is integrated recombination occurs at specific sites (between attB and attP sites in bacteria which both contain a spacer region and are flanked by integrase binding sites)
The prophage is normally excised from the chromosome by recombination between core O (spacer) sequences at attL and attR.
Sometimes it is excised aberrantly and some prophage ramins in the chromsome and the excised section carries bacterial DNA resulting in a mixed phage lysate.
The part of the bacterial DNA is scecifically those close to the att sites and not random.
67
What are transposable elements (aka transposons)
They are pieces of DNA that can move around the genome and insert at target sites via transposition.
They are found in all organisms and move to places in the genome, between chromosomal DNA and extrachromosomal elements like plasmids BUT ONLY WITHIN THE SAME CELL
Major source of mutation.
They move via two methods excision and integration OR by replication.
Excision and integration in a different location duplicating the target site due to DNA repair mechanisms.
Replication copies the DNA to from donor to target
Three types of transposon:
a) DNA only cut and paste elements have terminal inverted repeats and are widdespread. (only type found in bacteria)
They always contain an element bordered by short inverted repeats recognised by element encoded transposase. (bacterial carry other genes whereas eukaryotic usually only contains transposase.
bacterial DNA c&p elements that encode thei rown thansposition functions are called insertion sequences.
If a pair of these IS elements flank anther gene they can transpose the gene.
There can also be large Non composite IS element transposons which transpose genes as well
(B and C are eukaryote only)
b) Long terminal repeat (LTR) elements are retroviruses or similar and encode several proteins and reverse transcriptase.
C) Non-LTR elements can be autonomous or non-autonomous and encode proteins with a range of activities.
Long interspersed Elements (LINE) encode proteins for their own transposition (common in humans)
Short Interspersed Elements (SINE) do not encode proteins for movement and rely on LINE
45% of the genome is derived from Transposable elements, 7% of the DNA and 93% of the RNA.
Most move by cut and pase mechanisms and in bacteria only some move by nick and paste where the reansposon is still attatched to the donor DNA and is joined to a target.
a few transposons in bacteria can move by both.
This is essentially how the F plasmids integrates into bacterial chromosome.
The mechanism at the molecular level is:
1) Terminal inverted repeat DNA recognised by transposase
2) Transposases oligomerize bringing ends together
3) Transposon cleavage activated
4) cleaved transposon complex binds to target DNA (3' endd attacks DNA)
5)single strands are repaired by host
68
How does transcription take place?
Transcribes 5' to 3'
RNA polymerase recognises the promoter sequence and seperates the DNA
RNA is synthesised using DNA as a template.
Once RNA polymerase gets past the promoter a conformational change stabilises the interaction until the stop codon is reached.
69
Describe the structure and function of prokayrotic RNA polymerase and associated proteins
2 smaller alpha subunits (I and II)
2 Beta subunits creating a crabclaw shape (B and B')
And a small omega subunit on B'
There is also a seperate sigma factor that recognises the promoter and allows the RNA polymerase to bind.
the sigma factor is cashew shaped and has affinity to bind to both -10 and -35 bases before the promoter region and these regions are consensus sequences.
This only applies for primary sigma factors (housekeeping genes) and alternate sigma factors exist with alternate consensus sequences and different binding points.
The strength of a promoter and such the expression of the genes is determined by how similar the sequences are to the consensus (More similar = more frequently transcribed)
The combination of both is the holoenzyme
Other RNA polymerases share the general shape with different subunits.
70
Discuss the three eukaryotic RNA polymerases
RNA polymerase I:
In nucleolus
responsible for rRNA (ribosomal RNA)
RNA polymerase II:
In neucleoplasm
responsible for:
mRNA (messenger - protein coding)
snRNA (small nuclear NRAs found in spliceosomes)
miRNA (micro RNA - gene expression_
Rna polymerase III:
In nucleoplasm
responsible for:
5S rRNA (only large ribosomal subunit)
tRNA - places amino acids
snRNAs (same)
These RNA polymerases require transcription factors in order to be recruited to the start site (each polymerase has its own set).
71
What is the pre-initiation complex?
DNA sequences called the core promoted elements direct the assembly of the transcription initiation complex
TBP (in TFIID complex) binds to the TATA box promoting TFIIB to bind to BRE (B recognition element) sequence
The remainin gcompounds bind forming the pre-initation complex.
TBP is important for all the DNA polymerases.
72
Describe initation and elongation
RNA polymerases open about 14 bp of DNA (RNA pol II uniquely requires ATP through TFIIH helicase)
RNA polymerase often fails to make full length RNA the first time and aborts releasing RNAs of 2-9 nucleotides before creating a full elongated strand.
This occurs because the sigma factor has a loop in the active site that blocks RNA elongation and must be moved before abortive initiation can stop.
Displacement of the protein loop provies promoter clearance making RNA poly undergo a conformational change to associate it stably with DNA.
During elongation the DNA supercoils positivly infront of the Polymerase and negatively behind it requiring topisoerases to relieve tension.
In eukaryotes nucleosomes hinder RNA polymeraase and histone chaperonse remove them ahead of RNA pol and reassembles them behind.
73
How is mRNA processed after RNApol II activity with reference to elongation pausing?
Elongation is coupled to mRNA processing in eukaryotes
TFIIH phosphorylates the 5th serine on CTD region of the RNA plolymerase
This causes negative elongation factors to bind and pause transcription.
Phosphoylation rectuits RNA processing enzymes that add a guanosine cap to the 5' end of mRNA
Capping leads to phosphrylation of the second serine in the CTS and causes RNA pol II to resume elongation
74
How is transcription terminated
Prokaryotes:
Type I terminators (Rho-independent) require no additional factors for termination
Type II terminatiors (Rho-dependant) use Rho factor and ATP for termination.
Split 50/50 frequency
Allosteric model (eukarotes):
RNA poly II transcribes through polyadenylation and 3' end processing
RNA-processing complexes associate with the processing signals and phosphorylated CTD
mRNA is cleaved terminating transcription and releasing RNA pol II
Torpedo (eukaryotes)
mRNA cleaved at poly(A) site
RNA downstream of cleavage site is digested by ribonuclease (Rat1).
This dirupts polymerization and causes RNA pol II to dissociate from the DNA template.
75
How does RNA need to be processed before it is functional?
RNA cleavage is done by ribonucleases to ensure RNAs are the right length as rRNA and tRNA are usually synthesised as long precursor RNA
5' capping is the adittion of a guanine nucleotide to the 5' end of the mRNA via 5'-5' linkage. The guanine is methylated at N7 and only in higher eukaryotes at 2'-OH of the first (sometimes second) ribose
The 5' cap provides protection from degredation, stimulates translation of mRNA and aids transport from the nucleus to the cytoplasm.
The three steps of capping are:
1) Removal of the 5' phosphate (after 20-30 nt have been synthesised)
2) Guanylyl transferase uses GTP to attatch GMP
3) Guanine is methylated by a methyltransferase.
Polyadenylation of the 3' end (apoxmiately 200 adenosines on the tail end) catalysed by poly(A)polymerase. mRNA encoding metazoan histones have a different 3' structure.
RNA splicing:
RNA splicing is the removal of introns by transesterification reactions. In metazoa most introns are removed by the spliceosome but some self-splice.
Group I intron splicing is the 3'-OH of an exogenous guanosine nucleotide attacks the boundry of the exon 1 and ine intron detatching the intron and the 3'-OH of exon 1 attacks the boundry between exon 2 detatching them allowing for a transesterification reaction to rejoin the exons.
Group II intron splicing usies an internal adenosine 2'-OH to attack the exon 1-intron boundry and the 3'-OH of exon 1 attacks the boundry between exon 2 and the intron detatching them allowing for a transesterification reaction to rejoin the exons.
The plice site is detected by the presence of 5'-GU and 3'-AG
Intron boundaries are recognised by the splicesome.
The splicesome is comosed of 5 small nucleotide ribonucleoproteins (snRNPs) each of which contrains snRNA (100-300nt) and proteins.
The exon junction complex is left at splice junctions after splicing to mark the transcript as processing and to interact with export + transplation proteins preventing incompletely processed RNA from being exported.
76
What is alternative splicing?
The alteration of how pre-mRNA is spliced allows it to produce different versions of mRNA and therefore different proteins.
90% of human genes have alternate splice forms
Regulated by development and organ speficity.
77
How is RNA proessing coordinated?
RNA polymerase II coordinates capping, splicing and poly-adenylation.
Phoshporylation of the RNA pol CTD recruits capping enzymes and as elongation takes place it is further phosphorylated and RNA splicing machinery is recruited.
Adittional phosphorylation also leads to the recruitment of the complex that cleaves and polyadenylates the 3' end.
78
What is RNA editing and what are the mechanisms (in trypanosomes)?
the process that add/deletes bases from pre-mRNA or alters bases.
Produces an mRNA with bases that don't match DNA.
An extreme example is seen in the mitochondria of trypanosomes.
Uridine insertion and deletion in trypanosomes:
In insertion an endonuclease cuts mRNA at a mismatch and uridylyl transferase ads U's to the 3' end of 5' mRNA and RNA ligase ligates the fragments
In deletion an endonuclease cuts mRNA at a mimatch and removes the U from the mRNA before RNA ligase ligates the gap.
In humans substitution editing can take place such as in Apoplipoprotein B-100 where a C nucleotide is replaces with a U creating a stop codon resulting in a smaller protein (B-48) being produced.
79
What is RNA degredation?
RNAs have varying stability and have a half-life.
The determinants of RNA stability in eukaryotes are the 3' poly-A tail and the 5' cap structure.
RNA is degraded by exonucleases after the deadenylse-containing enzyme complex degrades the poly-A tail and on the 5' end when a decapping enzyme reoves the 5' cap.
Degredation is controlled by AU rich elements (AREs) which direct tail removal. ARE's are often found in mRNAs that are only required for a very short period of time.
RNA interference was discovered in C. elegans in 1998
in eukaryotes 2 types of small RNAs that allow targeted mRNA degredation are:
Short interfering RNAs (siRNAs) dreived from processing longer dsRNAs.
MicroRNAs (miRNAs) derived from RNA pol II transcripts
These form the RNA-induced silencing complex (RISC) and they bind to the miRISCs bin to 3'UTR of mRNA and silence genes repressing translation and activateing deadenylation, decapping and exonucleolytic degredation.
80
Why do cells regulate transcription?
To control quantity (how much of a specific protein they need)
To control timing (cell only needs certain proteins at certain times)
specificty (specific cell types need specific proteins)
In bacteria constitiutive or "housekeeping" genes are always expressed wherease regulated genes are only expressed under certain circumstances.
81
How do cells regulate transcription?
Poteins that contain a DNA binding domain and a transactivating domain can regulate transcription
10% of human genes encode transcription factors.
RNA poly holoenzyme can transcribe any gene with a functional promoter (repressors can disrupt the promoter region preventing transcription)
Targeted gene regulation is used most at transcription initiation, elongation or termination and the RNA itself can regulate
Proteins regulate transcription by binding to regulatory DNA sequences called operators
Enhancers: increase transcription
Silencers: decrease trancription
for example in the absence of DNA damage LexA represses the SOS genes (recA) and in the presence of DNA damage LexA is cleaved and SOS genes are produced to repair the damage.
82
What are operons and how is the E. coli lac operon regulated?
Operons are expression of genes that encode proteins that work together
In an operon genes adjacent to each other are transcribed together into one polycistronic mRNA that gets translated into seperate proteins.
The lac operon controls the metabolism of lactose to provide energy for E. coli.
The lac operon produces Beta galactosidase, permease and transacetylase.
B-galactosidase:
hydrolyses lactose to glucose and galactose
converts lactose into allolactose
Permease allows lactose to enter the cell
Transacetylase detoxifies the byproducts of lactose metabolism
the lac operon is repressed until lactose is present in the cell by lacl which is consitituively exppressed.
When lactose is present allolactose binds to lac represser altering its shape allowing transcription of the lac operon.
cAMP levels are increased by low glucose levels and vice versa.
CAP binds with cAMP and positively regulates the lac operon to be able to produce more glucose.
83
how is the trp operon regulated?
operons for anabolic pathways are turned off when the end product is readily avaliable
trp operon is turned off by excess tryptophan.
trpR codes an aporepressor (inactive repressor) and when trp is absent it doesn't bind to the trp operon
but when tyrptophan is present it binds and becomes an active trp repressor.
Transcription attenuation only produces short transcripts that do not encode structureal proteins based on the fact that transcription in bacteria is coupled to translation
Attentuation regulates amino acid biosynthesis in bacteria.
84
What are riboswitches?
They are segments of an RNA transcript that can bind to a small molecule that controls RNA secondary structure regulating transcription/translation
they have 2 regions (aptamer which binds the metabolite and expression platform which controlls transcription/translation)
The B. subtilis adenine riboswitch regulates adenine synthesis
85
How is transcription regulated by distal sequences?
Sequences located distal to the transcription start site can regulate transcription via enhancer or silencer sequences that contain a high density of binding sites for regulatory proteins
86
What are regulatory proteins in eukaryotes?
They are transcription factors that recognise specific DNA sequences.
They are often modular and have domains that:
DNA-binding domain
Activation or repression domain
Aid oligomerisation
Interact with other regulators
They often recruit other proteins that help regulate transcription but don't have DNA-binding activity themselves without the regulatory protein
87
How does histone modification affect transcription?
Histone proteins that comprise the octamer nucleosome core each have N-terminal tails taht can be covalently modified
Hyper and hypo acetylation can affect how tightly nucleosomes are packed by affecting lysine residues that bind strongly to DNA.
Hyperacetylation neutralises positively charged lysine residues and makes the nucleosome less compact and the DNA more accessible to trascription factors
Hyoacetylation increases histone-DNA affinity
Acetyl groups can directly recruit proteins via bromodomatin proteins which in turn can recruit other proteins that have an effect on transcription.
88
How do chromatin remodelling complexes affect transcription?
Binding sites for transcription factos can be inaccessible if wrapped around nucleosome.
Chromatin remodelling complexes can use ATP to move the histone octamer so that the region of DNA that is targeted by transcription factors and mechanisms is exposed between nucleosomes.
89
How does DNA methylation affect transcription?
DNA methylation affects chromatin structure.
in mammals cytosine residues at CpG sites are methylated
Transcriptional activation: Hypomethylation
Silencing: hypermethylation
methylation catalysed by DNA methyltransferase
methylation patterns are changed in: fragile X syndrome, cancer and aging.
Methylation and histone modifications affect each othe and methyl groups can recruit methyl-binding proteins which recruit other proteins that modify chromatin and affect gene expression.
90
What is the mediator complex?
It is a large protein complex of about 20 proteins (aprox 1 MDa) and RNApol II requires it to bind to the promoter
This was shown experimentally in vitro and RNAPol II wasn't initiated much at all without the mediator complex
91
How is transcription upregulated by external stimuli in eukaryotes (with examples)
External cues include: nutrient availability and stress (heat, genotoxic damage ect)
Horomones and cytokines are also important
Example 1:
for ELK1 in the absence of mitogens ELK1 binds to serum response factor but doesnt activate transcription. Mitogen-activated signal transduction pathways phosphorylate ELK1 which can then recruit mediator and promotore transcription of growth/proliferative promoting genes.
Example 2:
Activation of GAL genes in yeast:
Three genes encode enzymes that function sequentially to metabolise galactose and in the absence of galactose or presence of glucose then no transcription is present
only when galactose is present and glucose is absent are the GAL genes rapidly transcribed
This is regulated by Gal4 which binds to the UAS sequences activating transcription.
In the abesnce of galactose Gal80 bids to Gal4 and prevents transcription
In the presence of galactose Gal3 binds to Gal80 allowing Gal4 to do its thing recruiting SAGA and mediator to activate transcription.
Example 3:
Nuclear receptor proteins contain DNA-binding domain and ligand-binding domain
Ligand binding leads to a conformational change allowing them to drive transcription. (some cases ligand binding can allow translocation to nucleus)
Example 4:
Nutritional cues in yeast are responded to by Ume6
When Nitrogen and carbon sources are resnet Yme6 bind to DNA and recruits co-repressors (Sin3, Rpd3, lsw2)
Rpd3 is a histone deacetylase
Lsw2 is a nucleosome remodelling enzyme
these decrease transcription
In absence of these nutrients Ume6 is phosphorylated and doesn't bind resulting in Ime1 being recruited and acetylating histones increasing transcription.
92
What is the structure of the tRNA?
It is a cloverleaf structure with the anticodon in the anticodon loop (2nd loop)
There is also unusual modified bases in the loops
pseudouridine present in some bases in T loop (and before anticodon loop)
It is represented by psi
Dihydrouridine is present in the D loop and is represented by D
A CCA tail is added post-transcriptionally which is the site of amino acid attatchment
The first amino acid of a protein (methionine) has a specific tRNA molecule which is different to the tRNA used for elongation involving methionine.
93
Describe how tRNA is charged
Attatching tRNA to its cognate amino acid is called aminoacylation or charging
Carried out by aminoacyl-tRNA sythases using ATP as a cofactor producing aminoacyl-tRNA (or charged tRNA)
The reaction path is as follows:
The amino acid attatches to ATP on the first phosphate group displacing it
The amino acid is then transferred onto the terminal adenine of tRNA and AMP is released.
At least 20 different enzymes (one per AA) are used. Size exclusion makes sure only the right AA enteres the enzyme and when it does it is edited so that it fits in the second active site and the correct amino acid joins with AMP and the AA
94
What is the basis for the degeneracy of the genetic code?
Between the 1st base of the anticodon and the 3rd base of the codon other types of base pairs to watson-crick are allowed
C >G
A >U
U > A or G
G > U or C
I > U, c or A
95
Describe translation in prokaryotes
The first step and the rate limiting step is initiation and is controlled by the Shine-Dalgarno sequence (AGGAGGU) which binds to the anti-shine-dalgarno sequence on the 16S rRNA
The initiator tRNA (fMET-TRNA) is guided to the right place by IF1 and IF3 which protect both sides of the tRNA which also prevents the large rRNA subunit from binding
There are three sites that exist within the ribosome complex (A - aminoacyl site, P - Peptidyl site and E - exit site
after the initiatior the nect aminoacyl-tRNA molecule binds to the exposed codon in the A site in a complex with EFTu and GTP.
It then undergoes GTP hydrolysis and leaves the site
The peptide bond between AAs is catalysed on the ribosome by the peptidyl transferase centre. and peptidyltransferase is NOT A PROTEIN it is instead carried out by an RNA section of the 50S subunit and is known as a RIBOZYME.
EFG and GTP is used everytime it moves along a codon in the ribosome (peptidyl RNA moves from A to P site and uncharged moves from P to E to exit)
EFG is released and can be reused.
EFG is a structural mimic of EFTu:tRNA and binds to ribosome competitively forcing the movement of peptidyl-tRNA from the A to P site pushing deacylated tRNA to the exit site.
This process repeats extending the peptide chain
WHEN stop codon is reached termination happens
96
What is the differences between prokaryotic and eukaryotic translation?
Prokayrotes can have multiple proteins on the same mRNA and ribosomes can work heir way down one mRNA immediately after transcription.
Eukaryotes don't have the Shine Dalgarno sequence of mRNA and in mammals the RXXAUGG (Kozak sequence) is used.
They use the 7-methylG cap to assist in ribosome binding and the polyA tail stabilises the mRNA as such not all the mRNA acts as a template.
similarly to prokaryotes Initiation factor proteins (eIFs) are needed to assist translation start.
However the system is more complex with 12 eIFs grouped into 6 groups
The first step of initiation doesn't involve the ribosome at all and the mRNA is bound by the eIF4 family which recognises the cap and acts as a scaffold between the cap and polyA tail forming a closed loop complex. This complex shape is thought to be important for transcription
The pre-initiation complex scans for the start codon like in prokaryotes with integral GTPase (eIF2)
The eIF4A/4B complex act as an ATP-dependant helicase drawing mRNA through until the start codon reach the pre-initiation compex
When the start codon is identified the GTPase of eIF2 is activated causing conformational hift allowing the large subunit to bind releasing most remaining eIFs beside the 4E/4G complex that joins the cap and polyA tail.
Elongation in eukaryotes is ery similar to prokayotes but using equivalent elongation factors
eEF1A equivalent to EFTu (binds aminoacyl tRNA and GTP)
eEF1B equivalent to EFTs and exchanges GTP for GDP
eEF2 equivalent to EFG for ribosome translocation.
Termination is very similar in both as well.
But release factor eRF1 recognises all termination codons mimicing the structure of tRNA rather than prokaryotes which have to Release factors. Eukarote eRF1 also comes prebound with GTPase which is not present in prokaryotes.
The ribosome is recycled by breaking apart using ATPase ABCE1
97
How do protein synthesis inhibitors work and give examples
A range of compounds are shown to inhibit translation only on 70S ribosomes and not on 80S and is why they work as antibiotics without attacking human synthesis (side effects on mitochondrial 70S can occur)
kasugamycin blocks initiation
tetracycline blocks aminoacyl-tRNA binding
kirromycin blocks GDP -> GTP
aminoglycosides
chlorampheicol blocks peptidyl transfer
thiostrepton blocks translocation and erythomycin blocks transcloation at a later stage.
Fusidic acid is a narrow spectrum steroid antibioitc that inhibits elongation at the EG mediated translocation step.
Puromicin mimics aminoacyl-tRNA as it resembles and aromatic amino acid linked to a sugar-base and is treated by ribosome as if it were aminoacyl-tRNA adding the incorrect amino acid which doesn't bind to the protein and causes release. (double check textbook)
Ditheria toxin works as an antibiotic for eukarote cells as it catalyses the ADP-ribosylation of eEF-2 and blocks eukarotic protein syntheis and a single molecule is sufficient to kill a whole cell.
Ricin is a lectin protein found in the seeds of castor bean plants ricinus communis.
Very toxic by inhalation, injection or ingestion.
LD50 is 5-10ug/kg and 5-20 castor beans ingestion is lethal in adults.
Developed as a biological warfare agent in past (now schedule 1 agent) and involved in many incidents such as Georgi Markov assasination.
There is some interest in its theraputic usage as cancer therapy.
It works as a type 2 ribosome inactivating protein.
It contains chain A and B.
Chain B is a lectin glycoprotein that binds galactose and allows entry to the cell.
Chain A is an RNA N-glycosidase that depurinates a specific adenine of the 28S rRNA and one molecule can diable 50000 ribosomes.
98
What are the progressive changes in the formation of a malignant tumor?
A - Hyperplasia - cells divide more rapidly
B - Dysplasia - cells change form
C - Cells form a large mass (Carcinoma in situ) still in the same place (roughly stage 1)
D - Malignant tumor which can invade other normal tissue and enter blood and lymph systems (Diagnosis of cancer) (Stage 2 aand 3)
E - Metastases occurs and cancer takes root in other locations in the body. (stage 4)
99
What are driver and passenger mutations?
Driver gene mutations are mutations that directly or indirectly give the cell a growth advantage and essentially cause cancer
Passenger mutations are mutations that result from increased rate of division that has no effect on selective growth advantage.
Driver mutations can be positive regulators that promote cell proliferation or prevent cell death or
negative regulators which inhibit proliferation supressors or encourage cell death
743 genes with causal contributions to cancer as of feb 2024
100
How does the balance of growth and cell death affect division?
Normal growth is when prolferation > cell death
Repair is when proliferation = cell death
This can be changed via growth factos and growth-inhibiting factors.
101
What are the major events of the mitotic cell cycle, what are the main checkpoints
G0 / restriction point cells are not actively dividing
G1, S, G2, M and cytokinesis are main stages
G1 - growth before DNA replication
S - chromosome duplication
G2 - growh and prep for seperation of sister chromatids
M - mitosis (PMAT)
Checkpoints:
Restriction point checkpoint / G1/S - Is the environment favourable to start division
S phase checkpoint can inhibit further DNA replciation in S phase if something goes wrong.
G2/M checkpoint chacks if all DNA is replication and if the envrionment is suitable
Metaphase to anaphase checks all chromosomes are properlay attatched to spindle
Different cyclins are present during each phase of the cell cycle and they interact with Cdks to drive the cycle.
So progression through the cell cycle is dependent on
1) Cdk activity
2) Elimination of previous cell cycle stage proteins
Structure of checkpoint pathway:
Sensors bind to damaged DNA and activate transducers which launch damage response and this activates effectors which perform checkpoint functions. Prolonged arrest leads to apoptosis in many multicellular eukaryotes.
The sensor for spindle fiber defects is unknown but it is monitored and lack of attatchment is identified.
102
how is the cell cycle regulated?
It is regulated via growth factors and mitogens
These promote passage through the restriction point
Growth factors stimulate increase in cell size and are regulated by TOR kinase
Mitogens stimulate cell division by activating cyclin-Cdks
103
What are oncogenes?
They originate from proto-oncogenes that function in the growth signalling pathway to promote proliferation or inhibit apoptosis.
Oncogenes arise from genetic changes that either increase proliferation and apoptosiss inhibition or vice versa if the oncogenes promote apoptosis or inhibit proliferation.
Oncogenic mutations are genetically dominant so only a single allele mutation is required for cancer development.
They drive increased proliferation via:
Self-sufficiency in growth signalling
Ability to evade growth supressors
Apoptosis resistance
Replicative immortality.
There are six major functional classes of oncogenes that work in growth signalling or apoptosis:
Secreated growth factors/mitogens
Signal Transduction components
Transciption factos
Cell cycle regulators/drivers
Cell death inhibitors.
104
What tyes of muations can turn proto-oncogenes into oncogenes?
Three types:
Translocation/transposition of gene to a new locus under new regulatory control affecting expression
Gene amplification:
Multiple copies of the gene create multiple copies of the protein
Point mutations which create hyperactive or degredation resistant proteins.
105
What are exmaples of oncogenes for proliferation and cell signalling?
Mitogens and mitogen receptors:
Mitogens stimulate division by binding to a receptor
EGFR:
EGFR is mutatated in brain tumors breast cancers and ovarian cancers commonly
EGFR mutations include amplification of receptors lowereing the capture threshold of mitogens and deletion which results in treuncated receptor which trigger intracellular signalling in absence of mitogen.
Both of these promote proliferation by increased activation of signalling pathways
Ras protein (GTPase):
signalling protein which is activated by growth factor binding to receptor.
Inactive when bound to GDP and when mitogen binds to it it exchanges for GTP
Normal activation is transient
Mutation results in signalling pathway to become constitutively active regardless of mitogen presence.
Myc protein:
Acts in nucleus to stimulate cell growth and division
Mutations lead to increased expression and result in hundreds of copies of the normal oncogene
This can occur by:
Point mutations making it more stable and prevents degredation or
Translocation which results in increased gene expression in a different promoter region (in Burkitt's lymphoma it is translocated to antibody expression gene resulting in extreme expression.)
Cyclin D and Cdk4:
Cyclin D protein amplified in variety of cancers (oesophageal, bladder and breast) and results in unscheduled entry to S phase.
Cdk4 amplified in 12% gliomas and 11% sarcomas and drives progression through cell cycle start in absence of mitogen
106
What are exmples of oncogenes that affect apoptosis (cell survival and immortatlity)?
2 types of apoptosis:
Extrinsic triggered by extracellular signals binding to cell surface death receptors.
Intrinsic dependant on release into cutosol of mitochondrial protein normally in intermembrane space.
Intrinsic tightly regulated by Bcl2 protein family.
Increased levels of Bcl2 antiapoptopic proteins result in blockage of apoptosis creating immortal cells (cancer)
Increase of Bax induces apoptosis (opposite)
MDM2:
MDM2 regulates p53 levels which is required for damage checkpoint activation in absence of damage MDM2 ubiquitinates lysines in P53 C-terminal domain targeting it for degredation
In DNA damage both are phosphorylated allowing p53 to accumulate
MDM2 amplification common in adipose tissue tumors and soft tissue sacromas as it prevents accumulation of p53 in DNA damage.
107
How does telomerase mutation result in oncogene?
Normals cells cease division after 50-70 divisions (hayflick)
This is because telomerses get shorter with each division until they are too short.
Oncogenic mutations reactivate expression of telomerase enabling replicative immportality
Telomerase activity detected in all major cancer types.
108
What is the normal function of tumor supressor genes specifically retinoblastoma tumor supressor
Normal role is the inhibition of cell division via negative regulation or acivation of cell death.
Mutations can decrease activity loosing the inhibitory power.
Mutations are generally recessive so two mutations (two hits) are needed for a higher risk of tumors.
In retinoblastoma 87% of children with this condition worldwide die (mostly without proper screening)
There are 2 types:
Sporadic (60%) with single tumours in one eye
Herediatry (40%) with multiple tumors in both eyes
It is caused by 2 mutation events. In hereditary one mutation is inherited in the germinal cells and the other in somatic. In sporadic both mutations are in somatic.
This means that in hereditary individuals the RB tumor supressor gene is no longer heterozygous.
Both copies of RB tumour supressor need to be mutated to increase risk and this occurs when heterozygous parents produce a homozygous mutant recombinant.
The role of RB tumour supressor is the binding of E2F which activates genes required for G1/S transition.
109
Explain the role of p53 protein
The P53 tumor rupressor gene i the most commonly mutated gene in human cancer with ~50% of sporadic cancers having this mutation.
In Li-Fraumeni syndrome inheritance of a mutant p53 allele puts sufferers at 90% risk of developing a wide range of cancers.
P53 is an effector of DNA damage checkpoint and is normally degraded by MDM2 but in presence of DNA damage it is phosphorylated and tetramerises blocking nuclear export.
This leads to the arrest of the G1/S checkpoint preventing replication.
p53 also activates transcription for multiple other tumor supressor genes such as p21 (inhibits cyclin-dependant-kinases and arrests cells at G1/S) and GADD45 (Which binds to PCNA and blocks it from proceeding through S-phase replication check)
+ 14-3-3 which arrests at G2/M
+ Bax which is a positive regulator of apoptosis.
Failiure of checkpoints are major contributors to genetic instability which can activate oncogenes or lose more tumour supressor genes.
Most p53 mutations are recessive but some have a dominant negative effect and such one copy can disrupt normal p53 function (such as a bad subunit mutation that can disrupt their tetramer).
HPV produces E6 oncoprotein which binds to p53 targeting it for accumulation and E7 wwhich sequesters Rb disregulating E2F which is why HPV has a link to cervical cancer in women.
110
BIOL
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