Modules 1 & 2 Flashcards
What did frederick griffith do
Catalogued bacteria
2 viral strains - Griffiths
Rough small = Avirulent
Smooth large = Virulent
Transforming principle
Observation that an element of dead bacterial cells can transform avirulent bacterial cells into virulent
Griffith experiment
Treated rough strain with heat killed smooth strain, the new strain was now virulent
Avery-Mccarty-Mcleod experiment
Proved that DNA was the transforming component in the strain through adding enzymes such as DNAse to see which survived
Franklin & Wilkins
Used X-ray diffraction to identify a double helix structure
Chargraff
determined that base pairings had equal amounts of corresponding nucleotides
Conservative replication
Replication results in a molecule containing parental dna and a new strand
Semi conservative replication
Original DNA is split into 2 where one acts as a template for a new strand
Dispersive replication
Original DNA is chopped up and dispersed in a strand
Components of replication fork
- Helicase
- Pol E
- PCNA
- Pol A
- Pol D
- RPA
- RFC
Helicase function
Unwinds parental DNA using ATP
Pol E function
Replicative polymerase that extends leading strand
Pol D function
Replicative polymerase that extends the lagging strand, associates with PCNA
PCNA function
Acts as a clamp holding Pol E and D, stabilises them
Pol A function
Makes a complex with primase to synthesise primers for the lagging strand
RFC function
Clamp loader which opens the PCNA ring to enclose the DNA synthesised by Pol A
RPA function
binds to ss DNA to extend it , orientate it and protect it
Prokaryote replication
Has one point of origin, DNA unwinding site rich in AT
Eukaryote origin replication complex
Made of 6 proteins and loads CD factors, defines the orgins (pre-licensing)
What are replicons
Made of 2 replication forks which initiates synthesis
What is the initiation of DNA replication synchronised with
Cell cycle
What controls the cell cycle
Cyclins and cyclin dependant kinases
Cyclins and their roles
- B = block DNA synthesis
- D = activate G1/S cyclins
- E = restriction point
- A = Activates helicase
Transition to S phase
- Growth factor activates Cyc D and CDK4/6 (G1 cyclin)
- CDK phosphorylates pRB
- E2F TF s released which activates the transcription of S genes
- E2F upregulates G1/S cyclins
- G1/S CDK phosphorylate SIC1
- G1/S CDK is now active
- Cyclin A (S phase) phosphorylate helicase and loading factors in ORC
- ORC dissasociates
- CDC45 bind to helicase and activates it
- Pol A makes primers
mRNA processing (module 1)
- 5’ capping = protects mRNA from enzymatic degradation and aids with export
- processing of 3’ end = endonuclease creates a free 3’ - OH
- Polyadenylation of 3’ = Adds adenylic acid using Poly A and ATP
- Splicing = removes introns
Degenerative code
Not every codon codes for a unique amino acid
Translation initiation (module 1)
- EIF2 with GTP binds to met- tRNA
- 40s subunit binds to EIF1, 3 & 1A PIC forms
- EIF complex forms recognising the poly A binding protein & 5’ cap
- mRNA loop formed, activated
- PIC binds to activated mRNA
- Kozak sequence identifies the start site
- tRNA binds to start codon = GTP hydrolysis triggered
- 60s subunit binds
- EIF1A binds to A site and is then released if everything is correct
Translation elongation
- aminoacyl tRNA is delivered as a complex with EF1 A
- complementary codon and anticodon bind at the decoding site
- Ribosome proofs interaction
- GTP hydrolysis occurs releasing EF1 A
- tRNA in site A moves towards tRNA in site P
- amino acid in A binds to met tRNA
- EF2 binds and pushes mRNA and tRNA along using GTP
Translation termination
- ERF1 recognises stop codons
- ERF3 associates and hydrolyses GTP to release the polypeptide chain
- components of the ribosome are recycled
Causes of mutation
- UV
- Chemical modification
- Reactive oxygen species
- Cosmic radiation
- Errors in replication
Exouclease role
Proofreads in 3’ to 5’
Common mutations
- Spontaneous deamination of cytosine
- Benzylprene inserts itself into the double helix to disrupt it
- Thymine dimer formation causes a bulge in DNA
Direct reversal of damage
- Photolyase in plants uses visible light to reverse formation of thymine dimers
- Methyl guanine methyl transferase removes methyl from guanine in O6 position and moves it to its own active site (enzyme dies after)
Base excision repair
- DNA glycosylase removes incorrect base leaving a abasic site
- APE1 endonuclease cleaves abasic site
- DNA polymerase B fills gap with correct nucleotide
- DNA ligase binds new nucleotide
Mismatch repair
- MSH2 and MSH6 bind to mispaired segment
- MLH1/PMS2 binds
- DNA helicase unwinds DNA
- DNA exonuclease removes mismatched segment
- DNA polymerase and ligase repair section
Nucleotide excision repair
- XPC looks for lesions
- TF2H recruited as it has a helicase unit
- TF2H unwinds DNA using ATP
- RPA binds to form a protective bubble
- XPG & XPF act as endonuclease and cut damaged DNA
- DNA polymerase and ligase seal gap
2 methods for double strand breaks
- non homologous end joining
- Recombinatorial repair
Non homologous end joining
- KU80/70 heterodimer and DNAPK bind the ends of DNA together
- DNA ends are resectioned using artemis exonuclease
- DNA ligase ligates ends together
Recombinatorial repair
- 5’ exonuclease digests broken end of template strand creating a 3’ end
- unbroken daughter strand is ligated to the unreplicated portion of the parent chromosome
- RecA/Rad51 bind and cause strand invasion
- Loop of ss DNA created
- Holliday junction forms
- Crossover and ligation occur to generate a structure similar to replication fork
Primary structure protein
Sequence of amino acids
Secondary structure protein
3D shape
Tertiary structure protein
How secondary elements fold together
Quartenary structure protein
How proteins fit together
Adaptation stage CRISPR cas9
- bacteria identifies pathogen
- CAS 1 + 2 assemble and cut out a short protospacer
- Protospacer integrated into CAS 1+ 2 and becomes apart of CRISPR array
- Array now has the pathogen as a ‘memory’
crRNA maturation CRISPR Cas9
- Cas acts as nuclease and cleaves palindromic sequence in CRISPR array
- crRNA binds to CAS9
Interference CRISPR Cas9
- Mature crRNA acts as a template to attack pathogen
- crRNA recognises pathogenic DNA
- Cas9 acts as a nuclease to cut the DNA to make it unfunctional
Targeted cleavage by Cas9
- crRNA binds to tracrRNA to form a complex
- complex recognises incoming DNA
- PAM sequence binds to incoming DNA and aids with recognition for CAS9
- R loop forms forming 2 different nuclease active sites
- DNA is cut causing a double strand break
Cas9 structure
- Nuclease lobe = binds to target DNA
- Helical recognition lobe = binds to guide RNA
Role of RNA pol 1
transcribes pre r-RNA which is involved in ribosome components etc
Role of RNA pol 2
transcribes mRNA etc, involved in RNA splicing and post transcription gene control
Role of RNA pol 3
transcribes sort non coding RNAs such as tRNA
RNA pol 2 structure
- Unique transcription factors
- Differs in alpha subunits from pol 1 and 3
- Beta unit has a CTD which is the site for phosphorylation
essential features for RNA synthesis
- polymerisation is dependent on a template
- polymerisation is 5’ to 3’
- Watson crick base pairing
- Transcription requires a promoter to start
Stages in transcription
- Initiation
- Promoter clearance
- Limited polymerisation
- CTD phosphorylation
- Elongation
- Termination
Transcription initiation
- TF2D binds to TATA box in the minor groove which causes a bend
- TF2A and TF2B bind to the TF2D complex
- TF2B binds around the TAT box to stabilise
- TF2A crosslinks proteins to DNA
- TF2B recruits DNA pol 2 and TF2F
- TF2F acts as helicase to melt
- DNA pol 2 is stuck so TF2H has to come
- TF2H is docked via TF2E
- TF2H unwinds DNA infront of DNA pol 2
- TF2H phosphorylates CTD using ATP
- PIC open and promoter cleard
- RNA pol 2 undergoes confrontational change and releases TF2
- RNA pol 2 now interacts with elongation factors
pre - rRNA processing
- Nuclease cleavage via ribozyme
- Chemical modification = pseudoridylation and methylation
pre tRNA processing
- Trimming of 5’ end via ribozyme
- 3’ UUU replaced by CCA
-Anticodon loop reconstructed via splicing - Chemical modification
5’ capping (module 2)
- Gamma phosphate is hydrolysed via phosphohydrolase
- Guanine transferred from GTP to 5’ end via guanine transferase
- Guanine is methlated
Splicing (Module 2)
-U1 binds to 5’ splice site
- U2AF removes SF1 and allows U2 to bind to branch point
- U4-6 bind forming the spliceosome
- U6 unwinds from U4, U4 is removed
- U6 and U2 hydrolyse and have a conformational change activating the spliceosome
- 2 transesterification reactions take place
3’ Polyadenylation (Module 2)
- CPSF and CSTF bind to pre mRNA
- CF1 & 2 recruited and bind to CSTF
- Poly A polymerase binds to the 3 complexes
- CF1 & 2 cleave the mRNA
- PAP polymerises
- PABP stabilise the PAP
FG- Nucleoporin
- Involved in the transport of mRNP
- FGNP fill nuclear pore and interacts with mRNP allowing it to unfold
HIV
- Has a REV protein which binds to sequence elements on unspliced mRNA marking it as ‘spliced’ so the mRNP exporter transports the whole genome
Euchromatin
Decondensed
Heterochromatin
Condensed
Distal promoters/enhancers
- Distant from start site
- Upstream
- Mutations affect transcription but dont disrupt it
- Gene specific
Core promoter
- Present in every gene
- Vital to transcription
- Forms PIC
Ways to remodel chromatin
- Chemical modification
- Slide apart nucelosomes
- Pop histones in and out
- Modify DNA
Acetylation
- Decondenses chromatin
- Always associated with gene activation
- Reduces positive charge on histones to weaken interactions with DNA
Methylation
- Condenses chromatin
- Associated with long term gene repression
Mechanical modification of chromatin
- SWI/SNF bind to condensed chromatin
- Pushes nucleosomes apart using ATP
- Enhancers bind and recruit TF
- TF are enzymatic remodellers and pushes nucleosomes apart untill core promoter is exposed
- PIC assembles
- Mediator bridges TF with PIC
- ## Chromatin loop forms creating a signal for gene activation
Why was splicing maintained in evolution
- Helps create multi domain proteins
- Alterntive splicing can create many different proteins from a single gene
Cryptic vs Non cryptic intron
cryptic intron has an altered base making it have a decreased affinity at the branch site for U2 and needs a splicing activator
Sexual differentiation in flys summary
- sxl protein is only produced in females via casette splicing
- sxl repressors splicing in Tra protein in females, via alternative 3’ splice site
- Only functional Tra protein in females
- Tra protein binds to Rbp1 which activates splicing in dsx protein in females
- dsx protein = alternative poly (a) splicing
- dsx protein has a cryptic intron
role of dsx protein
dsx protein repress the transription of genes required for sexual differentiation in opposite sex
female sxl gene
acetylated which makes it automatically active
male sxl gene
methylated condenses and a translation stop codon gene which makes it inactive
translation repressor
- binds do eif4 complex
- displaces factors causing the mrna loop to open
- can also affect the mrna integrity and will make it cleaved
translation activator
enhance the efficiency and binding of eifs
Exosome pathways
- decapping = removes cap and eifs fall off causing loop to open
- deadenylation = shortens poly a tail, decreases PAPB which opens loop
- endonucleolytic = mrna is completely degraded
active vs dormant mRNA
- dormant mRNA has a short poly(a)tail which has maskin and cpeb making it stable
- active mRNA has a long poly(a)tail with initiation factors bound
dormant mRNA in pregnancy
- eggs have all dormant mRNA needed for stages after fertilisation
- when progesterone spikes it signals CPEB kinase
- kinase phosphorylates CPEB and maskin is released
- CPEB recruits PAP which extends poly(a)tail
- mRNA is now active
Ferritin role
iron quencher which decreases the availability of iron
TFR role
iron transporter
Low iron enviro
ferritin is down regulated and TFR is upregulated
IREBP is active and binds to IRE
High iron enviro
ferritin is up regulated and TFR is downregulated
IRE-BP undergoes a conformational change and cannot bind to mRNA
Regulator of iron
IRE-BP which is an allosteric protein
IRE-BP role
activator of TFR and repressor of ferritin
miRNA role
inhibits translation
siRNA role
inhibits translation and degrades the mRNA
RNA interference
- primary miRNA transcribed by RNA polymerase 2 forming a hairpin loop
- Drosha binds and cuts both strands at ss regions
- precursor RNA made
- Drosha dissociates
- Dicer binds and cuts at the loop at the ds region
- cuts made are uneven leaving a 2nt hangover
- RISC with argonaut binds to miRNA that will hybridise to mRNA
- miRNA will inhibit translation
- siRNA will cleave the mRNA
DSB repair mechanisms which result in the inclusion at a specific site
- Nonhomologous end joining
- Homology directed repair = uses a template with a point mutation to introduce it into the DNA which allows for targeted and specific genome edits
