Genome maintenance Flashcards
Role + structure of a promoter
Most genes regulated in groups (many diverse mechanisms)
Promoters define transcription start site + its direction.
Contains UP interacts w/ a-subunit in Pol, -35 TTGACA, -10 TATAAATG
RNA polymerase structure + how does it bind to promoters?
a - enzyme assembly, promoter recognition
β/β’ - catalytic centre, termination
σ - promoter recognition, binds both -10 & -35 box, gene switching
ω - maintenance of structure, assembly + recruitment of β
Co-operative binding to promoter - σ is only subunit that makes specific contact w/ DNA
αCTD interacts w/ UP element -> multiple contacts so affinity + specificity
What are the different σ factors?
70 - general (TTGACA - TATAAT)
32 & 24 - heat shock
54 - nitrogen (CTGGNA - TTGCA)
28 - flagellar (CTAAA - GCCGATAA)
All have different functions w/ different binding specificities.
- compete for RNA polymerase core enzyme to form holoenzyme
How are σ factors involved in gene switching?
Heat shock (42C) can induce σ32 which competes w/ σ70 for RNA pol.
Housekeeping genes turned down.
How is the phosphotransferase system and the CRP involved in catabolite repression?
RNA pol needs CRP which needs cAMP.
- cAMP synthesised by adenylate cyclase, activated by phos (PTS)
- PTS also involved in glucose uptake (glycolysis) -> prevents adenylate cyclase activation (low cAMP)
- when glucose exhausted, PTS activates adenylate cyclase
CRP is homodimer (helix-turn-helix), binds palindromic sequence upstream of promoter - interact w/ RNA Pol via a-subunit.
It bends DNA, 3 activation regions.
-> acts as universal control over many operons
CRP function in lac operon
Class 1
Bind polymerase via carboxy-terminal domain when bound to promoter, only binds if cAMP present.
Operon kept off by lac repressor tetramer binding O so promoter cannot be cleared
Unless lactose present - binds repressor forcing dissociation from O
SO lactose is an inducer + under neg feedback control as it is also substrate for B-galactosidase (lacZ)
CRP function in glap1 operon
Class 2
20 bases closer to polymerase sequence (too close) -> compensated by binding aCTD, aNTD + σ (multiple sites)
weak -35 sequence so CRP site overlaps
CRP function in AraBAD operon
Class III - arabinose metabolism, has 2 operators that bind AraC repressor + bends operon so Pol cant transcribe.
CRP presence interrupts repression by binding -90 sequence, bends DNA disrupting protein interaction (loop structure)
-> architectural factor
Arabinose binds AraC dimer + forces it off operon.
What is the structure + function of a 2 component signalling system?
Allows bacteria to respond quickly to env changes - N availability, heat shock, osmolarity
- Sensor kinase sits in cell membrane (receives input signal from env via input domain)
- Transmitter domain autophosphorylates His residue.
Receiver domain (Asp) phos. by His (Eukaryotes use Tyr or Ser) - Output domain (transcription regulator) -> binds DNA, associates Pol via a
- If receiver domain dephos. then activity blocked
glnALG operon function when inactivated
Aims to make glutamine from glutamate/oxoglutarate, when N is around.
Excess gln -> transcription of glnA initiates at Ap1, glnL/G at Lp (
->low level as blocked by NtrC - response regulator
NtrC is tetrameric protein, can bind DNA around Ap1/Lp + damp it down (weak promoter)
Never fully OFF, just runs at low level
glnALG operon function when activated
Low gln, NtrC phosphorylated + binds Ap2 via loop formation (ATPase needed), phos dependant.
Phos NtrC strongly activates Ap2 (σ54 not 70) -> strong transcription of gln synthetase (glnA)
+ strong Ap2 transcription reads through weak terminator to also transcribe low levels glnL/G
How is NtrC activated?
Regulated by NtrB which depends on PII (UMP4) - nitrogen sensor
Active - kinase that phos NtrC
Modified - phophatase that dephos NtrC via PII
at high gln/2oxo-glu ratio: UR/UTase nips off UMP from PII -> PII modifies NtrB
at low gln/2oxo-glu ration: UR/UTase adds 4xUTP to PII -> PII(UMP4), no NtrB interaction SO remains active
Summarise key features of the glnALG operon
Makes gln synthetase when gln:2oxo-glu ratio low.
Make sits own regulators: ntrB (sensor kinase) by glnL, ntrC (TF) by glnG.
It has a weak promoter after glnA gene.
NtrC must be phosphorylated to function fully (only activates σ54 promoter - Ap2)
Promoter selection according to alternate sigma factors controls operon activity.
- long range interaction, looping of intervening DNA between ntrC & σ54 to activate Ap2.
What is the SOS response?
Response to damaged DNA (stop & repair). SOS genes repressed by lexA.
Damage activates recA to become protease -> digests lexA, activating SOS genes.
When damage repaired, enough lexA to re-establish repression + inactivate recA remains for future damage.
SOS3, a calcium-binding protein, SOS2, a serine/threonine protein kinase, and SOS1, a plasma membrane-localized Na+/H+ antiporter
List the different networks of regulation
Operon - genes of same locus in metabolic pathways, compact co-ordinated regulation.
Regulon - networks of genes/operons regulated by same mechanism e.g. SOS
Stimulon - network of genes under control of 1 stimulus, can be via different mechanisms e.g. heat shock, N availability
Modulon - independent operons modulated via a common regulator e.g. catabolite repression (global network)
Rho independent transcription termination
Termination signal is GC rich followed by AT rich.
-> hyphenated dyad symmetry (palindromic)
Forms hairpin loop by intra-strand base pairing
- RNA Pol pauses
- DNA/RNA hybrid after hairpin is unstable
- RNA & polymerase dissociate
*RNA does the work
Function + structure of rho
Termination factor rho required at non U-rich termination sites.
Rho is hexamer (each subunit binds 12 bases) so hexamer binds mRNA when >72 bases exposed
Rho dependent transcription termination
Transcription + translation occur concurrently in prokaryotes. Ribosome dissociates at termination codon BUT RNA pol continues transcribing -> RNA sequence not coated w/ ribosomes.
Rho binds exposed mRNA & tracks along (uses ATP) to contact β subunit of RNA Pol.
- GC rich hairpin formed at termination site so Pol pauses
- RNA Pol dissociates
Trp operon
A, B, C, D & E code for enzymes needed for tryptophan synthesis.
In presence of tryptophan (co-repressor), trp operon repressed.
Active repressor binds operator sequence before leader + attenuator on operon.
products of operons repress operons but substrates for metabolism induce their operon e.g. lactose in Lac operon
What is attenuation?
- newly synthesised mRNA immediately associates w/ ribosomes
- transcriptional terminal is COUPLED to translation
- x10 effect on transcription
Depends on leader + attenuator sequence.
Attenuation causes premature termination of transcription after the leader at the attenuator IF tRNA-trp present.
- depends on mutually exclusive secondary structure formation
2 trp codon act as tRNA-trp sensor
Riboswitches
RNA sequences that can form complex secondary structures - interact w/ ligands + metabolites
- can control termination of transcription
- can control ribosome access to mRNA (translation
- cleavage (can acquire catalytic activity + cut mRNA for degradation)
Ligands include: amino acids, coenzymes, heat shock, uncharged tRNA, metabolites
How does microarray analysis work?
Used to monitor gene expression patterns.
mRNA from comparator samples:
a) 2 different cells (B & T cells)
b) 1 cell type in different conditions
Prepare + label w/ fluorescent tag (green & red), probes hybridised to DNA target sequences bound in know pattern (grid)
Samples then washed + fluorescence indicates bound probe quantity.
RNA seq provides state of the art alternative
Structure & function of enhancer region in class II regulatory gene
Binds specific TFs.
- has multiple binding sites for sequence specific TFs
- co-operate & interact w/ RNA Pol II
- position & orientation independent
-> gives cell specific regulation of transcription
Interact + influence activity of general TFs , provide signal for general TFs to move along mRNA
Structure & function of promoter region in class II regulatory gene
Binds general TFs.
- binding site for sequence specific TFs.
- most contain TATA element + initiator element (often AGT)
- position & orientation sensitive
TATA box is where TATA binding protein (TBP) binds to bring together all TFs.
Many genes have alternative promoters that are activated under specific conditions.
What are the roles of TBP?
TATA binding protein
- provides sequence specific protein-DNA interaction, bends DNA
- recruits other factors (TAFs) in TFIID to promoter region to help w/ transcription stimulation
- helps recruits TFIIA + TFIIB to determine positioning of RNA Pol
What are the roles of TFIIF & TFIIE in pol II initiation complex?
TFIIF:
- stabilises RNA pol interaction w/ TBP-TFIIB
- blocks non-specific binding of pol II to DNA
- attracts TFIIE & TFIIH
TFIIE:
- attracts + regulates TFIIH
What is the role of TFIIH in RNA pol II initiation complex (general TFs)?
- has ATP-dependent helicase to unwind DNA
- polymerase II C-terminal kinase activates pol + releases it from promoter
- has serines at pos 2 & 5 in eukaryotes which can be phosphorylated to activate polymerase.
How does promoter clearance by pol II occur?
- phosphorylation of of Pol II CTD by TFIIH
CTD consensus is 7 amino acids (ser5 phosphorylated) - TFII (D, A, B + E & H-kinase) dispensable for elongation.
- other proteins associate w/ TFIIH to form repairosome
^ defects in this function responsible for Xeroderma Pigmentosum
How is RNA synthesis co-ordinated by RNA pol II?
After promoter clearance, pol II pauses so capping complex is recruited to prevent RNA degradation.
Elongation may abort or progress transcription (CTD additionally phosphorylated at ser2 by CDK9)
How is transcription terminated?
RNA pol II arrives at poly-A signal (PAS), 3’-TTATTT-5’ -> adds 5’-AAUAAA-3’ to mRNA
Cleavage site 15-30nt downstream of the PAS.
RNA pol II adds A bases to 3’ end protecting it
More efficient if also a U-rich upstream signal element (USE) + GU-rich downstream signal element (GSE)
What is the electrophoretic mobility shift assay used for ?
Consensus sequence analysis if you know the DNA sequence.
- purify protein + add to known DNA sequence that has been 5’ end radiolabelled
- run protein-DNA mix on native gel
- DNA bound to protein will be in higher mol weight complex + run slower in gel
What do specific transcription factors (TFs) bind to?
DNA sequences in promoters and enhancers of a gene.
Have a modular structure.
How many superfamilies of DNA-binding domains are there?
4 superfamilies
What types of domains can transcription factors have?
- Activation domains
- Repression domains
- Domains to interact with other TFs
What are the two categories of transcription factor structures based on their configuration?
- Monomers
- Dimers
What is an example of a monomeric DNA-binding domain?
Helix-Turn-Helix (HTH) e.g. homeodomain (HD)
Zinc finger (ZF)
What is an example of a dimeric DNA-binding domain?
Basic-Leucine-Zipper (bZIP)
Basic-helix-loop-helix (bHLH)
What is the Helix turn-helix (HH) motif?
A structural motif where Helix 3 binds the major groove and Helices 1 & 2 stabilize the structure. The N-terminal arm contacts the minor groove.
Example includes the homeodomain family.
How does Zif-268 interact with DNA?
Three Class 1 zinc fingers bind DNA, with amino acids within the fingers generating binding specificity.
This specificity is critical for transcription factor function.
What is the significance of C4 zinc fingers?
Found within nuclear hormone receptor family.
They are a large superfamily of ligand-dependent transcription factors and receptors.
Examples include nuclear estrogen receptors ERα and ERβ.
Can nuclear estrogen receptors bind DNA as homodimers?
Yes, they can bind as homodimers (αα or ββ) or as heterodimers (αβ).
This flexibility allows for diverse regulatory mechanisms.
What is a basic-helix-loop-helix?
Similar structure to bZip.
H-L-H domains interact, so basic regions can then bind DNA + target DNA sequence CAnnTG.
Many are heterodimers that use a common type-1 partner.
There is DNA specificity due to type-2 variable partner.
Examples of bHLHs in muscles and neurons
Muscle - MyoD, activates genes to make muscle cells
Neurons - Atoh1, activates genes to make neuronal cells
What is the role of CLOCK-BMAL1 in gene transcription?
CLOCK-BMAL1 (helix loop helix heterodimer) binds E-box enhancer sequences in circadian genes (CACGTG)
This binding stimulates the transcription of circadian genes.
What type of domain does CLK possess?
Gln transactivation domain. PolyQ interacts w/ general factors.
Pol II stimulated to transcribe gene
What are the components of a nucleosome?
1 linker histone (H1) and 8 core histones (2 of each: H2A, H2B, H3, H4)
Central H3-H4 tetramer which 2xH2A-H2B dimers then bind.
Nucleosomes consist of DNA wrapped around these histones.
How many turns of DNA are wrapped around each histone in a nucleosome?
2 turns of DNA. Packaged into 3 nucleosome wide structure ~30nm
This structure is crucial for DNA packaging in the nucleus.
What forms the histone fold-domain in histones?
Central helix is histone fold domain - where histones bind each other.
e.g. H2A-H2B dimer binds H3-H4
N terminal tails exposed + have highly basic residues -> modified for chromatin remodelling.
These helices allow histones to bind to one another.
What are the various post-translational modifications of histone tails?
- Methylation of Lysine (K) and Arginine (R)
- Acetylation of Lysine (K)
- Phosphorylation of Serine (S), Threonine (T), and Tyrosine (Y)
- Citrullination of Arginine (R)
- Ubiquitination of Lysine (K)
- Sumoylation of Lysine (K)
Post-translational modifications significantly influence chromatin structure and gene expression.
Which modifications are primarily focused on in transcription?
- Acetylation of Ks in H2A, H2B, H3 & H4
- Methylation of Ks & Rs in H3 + H4
Acetylation and methylation play crucial roles in regulating transcriptional activity.
Why is histone modification so complex?
Occurs in situ (whilst DNA bound) + correlates w/ functional status of relevant genomic locus.
Changes define epigenetic ‘histone code’ -> defines binding potential of chromatin associated factors.
e.g. H3-K18 can be trimethylated + acetylated
The histone code is a set of modifications that influences gene expression and chromatin structure.
What is the role of histone acetyltransferases (HATS)?
HATS add acetyl groups (CO-CH3) to histones
Examples include yeast Gcn5, CBP, PCAF, and TAF250.
What is the function of histone deacetylases (HDACs)?
HDACs remove acetyl groups from histones
Examples include yeast Rpd3 and human HDAC1.
What is the effect of acetylation on transcription?
Acetylation is associated with transcriptional activation
It reduces charge, leading to less cross-binding of histone proteins and a relaxed chromatin state.
What is the function of CLOCK in chromatin organization?
CLOCK has HAT-activity, acetylating H3 and H4, which opens up the core tetramer for DNA accessibility
It also relaxes chromatin and recruits TFI and Pol-II. Keeps basal promoter region open for TFII + Pol-II
What is the significance of p300/CBP in transcription?
p300/CBP can interact with various transcriptional regulators like pCAF (HAT)& TBP.
Acteylation by p300/CBP & TAFII250 facilitates the assembly of the pre-initiation complex -> driving transcription.
It helps drive transcription by recognizing the promoter TATAA.
Fill in the blank: Histone acetylation by _______ facilitates assembly of the pre-initiation complex.
p300/CBP & TAFII250
What are lysine methyltransferases (KMTs)?
Enzymes that add methyl groups to lysine residues
What do lysine demethylases (KDMs) do?
Remove methyl groups from lysine residues
What is associated with lysine hyper-methylation (me2/3)?
Transcriptional repression
1, 2 or 3 methyl groups can be added to NH3+ residues on Lys. Replaces P
Which human KMTs methylate histone H3 on Lys 9 causing H3K9m2/3?
SUV39H1 and SUV39H2
What do Protein Arginine Methyltransferases (PRMTs) catalyze?
Transfer of methyl groups from S-adenosylmethionine (AdoMet) to arginine residues
How many Protein Arginine Demethylases are there in humans?
Only one, JMJD6
What is arginine methylation usually associated with?
Transcriptional activation
e.g. PRMT1 methylates H4R3
What does PRMT4 methylate, and what is its effect?
Methylates H3R17 + non-hisotne protein p300/CBP triggering its HAT activity for transcription
What do modified histone tails alter?
Chromatin function by driving association with specific bound regulatory proteins
For example, H3K4me3 & H3K9me3 bind to PHD finger domain proteins like ING2 and BPTF.
Which proteins bind to H3K9me2/3 & H3K27me2/3?
Chromodomains - HP1
These modifications are associated with heterochromatin formation.
What do General-Kac & H4K16ac bind to?
Bromodomains - PCAF
This binding is significant for chromatin acetylation and gene activation.
What is the role of remodeling complexes?
Controls transition between chromatin states.
They speed up histone sliding to reveal DNA binding sequence -> TFs can recruit HATs to fix chromatin in active state.
They include ATP-dependent chromatin remodeling and histone acetylase/deacetylase containing complexes.
Name the two major classes of chromatin remodeling complexes.
- ATP dependent chromatin remodeling histone sliding (e.g., SWI/SNF, NURF)
- Histone acetylase/deacetylase containing complexes (e.g., SAGA, PCAF)
These complexes facilitate the dynamic changes in chromatin structure.
What is euchromatin associated with?
Active gene expression
Euchromatin is less condensed and accessible for transcription.
What characterizes heterochromatin?
Generally inactive
Heterochromatin is more condensed and less accessible to transcription machinery.
What does FACT do during transcriptional elongation?
Removes H2A-H2B dimers ahead of polymerase -> histones disassemble
This allows Pol II to advance and transcribe the DNA.
What happens once Pol II has passed during transcriptional elongation?
FACT helps reassemble histones to form new nucleosomes further upstream, towards 5’ end
This process is crucial for restoring chromatin structure after transcription.
What are enhancers in gene regulation?
Enhancers are regulatory elements that work at a distance to activate transcription by facilitating the recruitment of the transcription pre-initiation complex
They enable DNA looping and drive efficiency in forming the pre-initiation complex.
What are the three common types of activation domains?
- Proline-rich
- Glutamine-rich
- Acidic (glutamate + aspartate rich)
These domains interact with other proteins to activate transcription.
What role do Locus Control Regions (LCRs) play in gene regulation?
LCRs act like enhancers but function over longer distances and have multiple binding sites for TF complexes to activate multiple linked genes
12 LCRs have been identified in humans so far.
What is the function of insulators in chromatin?
Insulators provide boundaries for domains within euchromatin and prevent unwanted interactions between chromatin regions
They stop enhancers from regulating incorrect genes.
What is the difference between barriers and insulators?
Barriers are more important at the boundary of heterochromatin and euchromatin, preventing heterochromatin from spreading into gene-containing regions
Insulators primarily define interactions within euchromatin.
What are nucleosomes?
Nucleosomes are fundamental structural units of chromatin
They consist of DNA wrapped around histone proteins.
What is the structural organization of chromatin in the mammalian nucleus?
Chromatin folds and loops to form discrete chromosome territories, with euchromatin and heterochromatin occupying different zones as CTs are polarised.
This organization is crucial for gene regulation and expression.
True or False: Core promoters are the same as enhancers.
False
Core promoters are distinct regions that initiate transcription, while enhancers are regulatory elements that enhance transcription from a distance.
Which parts of the histone can be modified?
N-terminal tails which are exposed + have highly charged basic residues.
- essential for chromatin remodelling
What are higher order domains?
Genome regions can be gene rich or poor. Different chromatin domains fold into distinct 1Mbp DNA foci in situ.
Organise eukaryotic chromosomes - compact chronatin fibers.
active + inactuve hgiher order domains separated
How were the sites of RNA synthesis discovered?
Used Br-UTP (green)
Found mRNA synthesis sutes clustered in ‘factories’ that have many genes w/ 5-10 active RNA pol-II complexes
Give an example of co-localised transcription
3 NF-kb target genes in same region in HUVEC cells following TNF treatment.
Co-localised + recruitment is ordered
tertiary trasncription complex provides synergistic co-regulation
Give an overview of how gene expression is controlled by signal transduction (gradient)
- Secreted ligands (morphogens) released from a source (organiser) signal to nucleus to drive tissue specific signalling pathways
- Extracellular morphogens lead to intracellular TF gradients that drive alternative transcriptomes in cells
- TFs function at different thresholds to regulate genes via a digital (OFF-ON) and/or analogue (GRADED) level to drive cells to different fates.
How does extrinsic signalling occur?
Involves transmembrane or nuclear receptors.
Ligand binds receptor + either:
- causes change in conformation -> signal transduction via phosphorylated 2nd messenger (becomes TF)
- ligand is steroid hormone so is internalised at nucleus (becomes TF)
How does intrinsic signalling occur?
TFs either increase or decrease transcription - driven by basal machinery.
One TF can drive multiple genes to be upregulated -> cascade
How does a transcription cascade work?
Master TF preferentially recognises 5’-ATTTAAAT-3’ in enhancer regions. MTF can bind 5 similar sequences present in 20 genes.
Primary genes (A1, B6, C11)-> TF proteins
Secondary genes indirectly transcribed -> TF proteins
e.g. secondary protein D21 is an activator of tertairy genes (G41-45)
What determines a cells fate
Extracellular conecnetration of morphogen.
Intracellular MTF conc dircetly correlates w/ conc of morphegen received.
So cells nearest morphogen source generate most MTF - due to graded level of signal transduction factor (morphogen).
MTF conc leads to differential transcriptomes & alternative cell fates.
A single TF can drive alternative transcriptomes at different concs.
Digital function in a MTF
Will change the cell if it is present above a threshold.
e.g. any cell w/ > 25nM MTF will convert from fate X to Y
Switch occurs as at the threshold, gene Y can activate (auto-regulate) its own expression.
Once upregulated, cell has Y-competence so Y fate will persist even if MTF removed.
Analogue function in MTF
Morphogen gradient cna also provide an anlogue signal to sub-pattern Y cells.
e.g. 25nM = Ya, 30nM = Yb, 35nM = Yc
at low concs, only highest affinity targets bound by MTF
at high cocns, MTF activates lowest affinity sites, excess mols can bind further sites,
higher the ceoncnetration, more genes bound so much ahrder to spot lower affinity genes
Repression in a transcription cascade
lower affinity genes often codes for repressor for a higher affinity gene (e.g. B7 for A genes)
Single TF can generate cells w/ very different transcriptomes due to alternate casacde triggered
BUT different fate of low affinity gene could be due to expression of additional genes
How do patterning & segmentation provide AP identity?
Segmentation genes provide AP identity within each individual segment (polarity)
Patterning genes provide unique segment identity along AP axis
Patterning driven by initial maternal bicoid mRNA concentration gradient.
What genes are expressed in AP patterning in drosophila development?
- high conc bicoid protein activates zygotic hunchback (anterior)
- hunchback gradient est domains exprssing distinct GAP genes
- hunchback combines w/ GAP genes to co-activate pair-rule genes
GAP genes - kniros, tailless, giant, knippel
pair rule genes - even-skip
Pair rule gens activate segementation genes (wingless/hedgehog)
hunchback, GAP + Pair rule TFs combine to co-activate patterning genes (Hox)
Discovery of Hox genes
Ubx mutant cuases T3 to turn into T2 - complete w/ wings instead of halteres.
-> master AP-patterning genes are homeobox (Hox) family
Same in mice but they have multiple Hox genes
Lewis in 1970s
All patterning genes driven by concentration of retinoic acid
How is pluripotency generated from transcription cascades?
Initial signalling generates broad fate restriction.
e.g. endoderm, mesoderm, ectoderm + neuroectoderm.
Gradients then re-used to generate sub-fates -> depends on precise time, conc + order in which it receives key morphogens
In vertebrates, what is a key factor that drives development?
Competition between 4 morphogen gradients - TGF, BMP, FGF & WNT.
-> determine swhich MTFs will drive transcriptome
these 4 pathways + their interactions generate all possible trasncriptome combinations -> all diverse cell types
How is prokaryotic DNA replication initiated?
In most prokaryotes, factors bind single specific OriC site to form replication bubble.
Easily unwound DNA is A-T rich (weaker) - broken due to mechanical twisting of DNA by DnaA
- DnaB (helicase) is loaded + binds both strands by DnaC (helicase loader)
- primase can use DnaB as reference point for specific pairing
DNA synthesis semi-conservative (Meselson & Stahl) - parent strand used as template.
Chain elongation in prokaryotes
dNTPs have triphopshate + hydroxyl end - nucleotide monophosphate added to chain w/ release of diphosphate.
Synthesis only in 5’->3’ direction
Why is replication semi-discontinuous?
2 growing forks at once in ‘replication bubble’:
Leading strand (5’->3’) is continuous
Laggin strand (3’ -> 5’) is discontinuous, added in steps
Okazaki fragment synthesis in prokaryotes
- Primase synthesises short RNA primer
- DNA Pol III extends primer into Okazaki fragment
- DNA Pol I can displace RNA primer in the way (5’->3’ exonuclease activity) + add bases
- DNA ligase joins adjacent nucleotides w/ phosphodiester bond
some DNA polymerases have 3’->5’ exonuclease activity (proofreading)
DNA Pol III holoenzyme structure + function
Dimeric - has monomers associated w/ leading + lagging strand.
- a-subunit is polymerase centre
- clamp loader grabs beta subunit which recognises DNA/RNA hybrid, csliding clamp locked over primer
- a-subunit binds clamp + synthesisies strand
exonuclease activity in palm shape area of a-subunit
DnaB (helicase) structure + function
Hexameric - catalyses unwinding of DNA duplex to expose single strand DNA
- Different helicases w/ different polarities on 2 strand in duplex
- ATP hydrolysis provides energy for unwinding
SSB structure + function
Single stranded protein - tetrameric
Binds ssDNA to prevent duplex reannealing - >1000 fold afffinity for ssDNA over dsDNA
Lowers DNA melting temp promoting denaturation
Exhibits co-operative binding.
DnaG (primase)
Initiates okazaki synthesis on ssDNA template
- displaced by DNA Pol III after 10-12 ribonucleotides added
- Pol III synthesises from 3’OH groups on RNA primer
Forms complex w/ helicase in lagging strand synthesis
DNA Pol I
Lagging strand synthesis - 5’->3’, catalyses chain elongation by formation of phosphodiester bonds
- only extends chains from 3’OH termini, cannot initiate synthesisi of new chains
- primer removal in lagging strand synthesis
has both 3’ (proofreading) and 5’ (primer excision) exonuclease activity
How is bacterial replication terminated?
180 degrees from OriC are Ter sites (replication fork traps)
- 2 sets for both directions of DNA synthesis
- - binding sites for Tus (DNA binding protein) as a monomer
Can also directly inhibit replication fork progression by contacting DnaB (inhibiting unwinding)
Okazaki fragment synthesis in eukaryotes
- RNA primer extended by DNA pol alpha
- Pol delta + helicase push aside primer to extend strand
- Fen1 (endonuclease) cuts at branchpoint /flap at 5’ end DNA
- DNA ligase links adjacent fragments
What is the replicon?
DNA between adjacent termini, can replicate independently with its own origin site.
Almost all replication units are bidirectional.
Eukaryotes have fragmented genome (multi-chromosomal) so multiple units of DNA replication needed
Initiating DNA synthesis in eukaryotes?
Origin defined by complex 3D combination of chromatin features that generate ORCs (origin recognition complexes)
origin function is redundant - more origins than necessary -> stochastic component to origin selection
- no need for specific sequence
ORCs give platform for assembly of pre-initiation complex - recuited in cell-cycle dependent manner (controlled by cyclin/CDK)
-> proteins only bind ORCs in window of opportunity to form multi-complex
What organism could be described as a paradigm of origni function?
S. cerevisiae - 1st replication origins to be characterised.
Discovered autonomous replicating sequences (ARS) - contains 3 essential conensus domains (A had high T-A)
- bound by ORC complex
Which cyclins contribute to origin timing?
CLB6 - acts in early S-phase, rapidly degrades
CLB5 - operates late in S phase
*in yeast
each origin has an innate timing preference
Competency in yeast DNA replication
Competency is lieklihood of ARS being used to replicate DNA
Some ARS elements very strong, others very weak
Origins for human DNA replication
AT rich sequences w/ no clear consensus - defined by local chromatin context.
What is the Mcm complex composed of?
- Cdc6 - highly unstable, synthesised during G1, binds ORC + recruits Cdt1
- Cdt1 also only expressed early in G1 - it loads McM to establish pre-RC complexes
BUT
Geminin expressed late G1 + S phase - blocks Mcm loading
If factors are loaded, then that region is licensed to duplicate DNA during that cell cycle
S pahse programme in mammalian cells
Takes 9-10 hours
- Euchromatin is replicated 1st
- Heterchromatin replicated last
High repeat regions at centrosomes + telomeres also replicate last
How can we study genome wide replication timing?
lFlwo cytometry - cels from S phase fractions given BrdU (thymidine analogue)
- anti-BrdU antibody with a fluorescent dye used to detect
Immunopurified then sequenced to profile replication regions.
If sequence replicated durig specific period - copy number will double in that sample
Doesnt all happen at once, some early/late
How does CDK & DDK regulate initiation of pre-RC complex?
When CDK + DDK high at onset of S-phase, initiation of replication can occur.
Cdc45, Sld & GINS protein recruit replisome to initiate synthesis
How is DNA replication controlled under stress?
Replication stress - causes DNA damage, potentially mutagenic
RPA prevents DNA looping on itself by binding to exposed ssDNA
PARP1 adds ribose residues, acts as tags for checkpoint kinase 1 (Chk1) to bind
- activated Chk1 activates p53, S phase checkpoint activated
- Cdk inhibtitors activated (p21, p27, Cdc25)
- p21 + p27 block pregression to S-phase
- Cdc25 blocks progression during S phase to allow repair (stalling mechanism)
Repair proteins interact w/ stalled replisome at fork (PbR)
What happens if the replication fork collapses?
Dormant origins (backups) activated.
- Mcm2-7 complexes displaced during initiation move along DNA to provide helicase function (unwind DNA)
Beltand braces mechanism - reduces likelihood of errors
MCM2-7 genes are prevalent in squamous cell carcinomas - when mutated, shown to causes cancer in mice.
How is the nucleosome maintained?
mRNA that encodes histone normally very unstable
Histone genes upregulated in S-phase (50-fold increase in mRNA)
- timed by cell cycle
How are histones produced accurately for the replicated DNA?
- H2A-H2B dimers released + held by histone chaperones, H3-H4 tetramers remain bound but rrandomly assigned to leading/lagging strand.
- DNA Pol III duplicates each strand + chaperons randomly isnert H2A-H2B
- new histone proteins intermix w/ parental histone (double toal mass)
- histone methylase complexes recognise N-terminal methylation on parental + copy it to new histones locally (read write mechanism)
What are the 3 groups of mutations?
- Point mutations
- Large scale INDELs
- Chromosomal rearrangements
What is a point mutation?
INDELs only one DNA base
What are large scale INDELs?
Repetitive sequences, microsatellites
- occur rapidly and can distinguish I dividuals based on number of copies.
What are chromosomal rearrangements?
Dramatic impact on organism function
What are the two sources of small scale point mutations?
- Inaccuracy in DNA replication
- Damage to DNA
What role do mutations play in biodiversity?
Biodiversity exists as a result of mutations
What is a silent mutation?
Single base pair change in DNA but no resulting amino acids change
What is a missense mutation?
DNA change results in changed amino acid sequence
What is a nonsense mutation?
DNA change encodes premature stop codon
- almost always deleterious
How often does DNA Pol make a mistake?
1 mistake for every 10 copied base-pairs
What are the two mechanisms of error correction in DNA replication?
- Proofreading
- Mismatch repair
How does proofreading work?
DNA Pol detects mistake via kink in DNA, slows down, exonuclease removes last added base
Reduces error rate 100 fold
What is mismatch repair?
Corrects errors that escaped proofreading before permanent damage
What is the role of the Muts protein in mismatch repair?
Scans genome to find kinks and makes them more pronounced
What does MutH do in the mismatch repair process?
Excises one strand near mismatch site
What is the function of helicase in mismatch repair?
Unwinds DNA from the nick towards the mismatch
What fills the hole left after mismatch repair?
DNA Pol fills hole up with correct base-pairs
What connects adjacent base pairs after mismatch repair?
DNA ligase
How does mismatch repair know which strand to remove?
methyl directed MMR
MutH recognises strand to excise due to Dam methylase:
- methylates DNA every 5’-GATC-3’ on A, both strands
- after replication, DNA hemi-methylated (only 1 strand carrying CH3)
- MutH sits on methylated sites + nicks only uumethylated DNA (new strands)
Distinguishes mutated vs original strands
likely evolved form canonical repair system - MutL has function of both MutL & MutH
What is DNA damage caused by?
Inaccuracy in DNA replication, Damage to DNA, Spontaneous damage
DNA damage can occur through various mechanisms, including errors during replication and external factors.
What is spontaneous DNA damage?
Occurs due to most chemical reactions in biology being reversible
This means that the rates of reactions are not equal in both directions, leading to potential damage.
What is an example of spontaneous DNA damage?
Deamination of cytosine makes uracil
This process alters the base composition of DNA.
What happens during depurination of guanine?
It makes apurinic deoxyribose, a sugar without a base
This type of damage results in the loss of a purine base.
What does deamination of methylated cytosine produce?
Thymine
This process can lead to mutations in DNA.
What is alkylation in the context of DNA damage?
Transfer of methyl/ethyl groups to reactive sites on bases or to phosphates in DNA backbone
It can lead to wrong base pairing, such as thymine instead of cytosine in the case of 0-methylguanine.
What is the effect of oxidation on DNA?
Reactive oxygen species can cause mutations, such as OxG wrongly base pairing with adenine
This is the most common mutation in cancer unless the organism is in an anaerobic environment.
What wavelength of radiation is particularly dangerous to DNA?
260nm wavelength (UV radiation)
It leads to the fusion of two pyrimidines, such as thymine dimers, preventing base pairing and stopping polymerase during replication.
What test is used to determine if a chemical is mutagenic?
Ames test
This test assesses the mutagenic potential of chemical compounds.
What type of radiation causes double-stranded breaks in DNA?
Gamma radiation and X-rays
These breaks are difficult to repair as there is no reference/template strand.
What mutation occurs when a gene in Salmonella is knocked out?
Prevents histidine synthesis
This leads to a single point nonsense or frameshift mutation that converts his+ to his-.
Fill in the blank: ______ can lead to wrong base pairing in DNA.
[Alkylation]
What is alkylation in the context of DNA damage?
Transfer of methyl/ethyl groups to reactive sites on bases or to phosphates in DNA backbone
It can lead to wrong base pairing, such as thymine instead of cytosine in the case of 0-methylguanine.
What is the effect of oxidation on DNA?
Reactive oxygen species can cause mutations, such as OxG wrongly base pairing with adenine
This is the most common mutation in cancer unless the organism is in an anaerobic environment.
What wavelength of radiation is particularly dangerous to DNA?
260nm wavelength (UV radiation)
It leads to the fusion of two pyrimidines, such as thymine dimers, preventing base pairing and stopping polymerase during replication.
What test is used to determine if a chemical is mutagenic?
Ames test
This test assesses the mutagenic potential of chemical compounds.
What type of radiation causes double-stranded breaks in DNA?
Gamma radiation and X-rays
These breaks are difficult to repair as there is no reference/template strand.
Describe the Ames test in Salmonella
hisA gene in Salmonella knocked out so prevents histidine synthesis.
- single point/nonsense converts his + to his-
Reversal mutation in presence of mutagen converts his - back to his+
Grown on media w limited hisitidine
Not a yes or no result - rough, indicative
Variability between humans and Salmonella
What is Junk DNa?
Fastest changing DNA, not under stabilising selection so drifts and acquires mutations
E.g. pseudogenes now don’t have function but could in future
What are the 4 general mechanisms opf DNA repair?
Direct reversal DNA damage
Base excision repair
Nucleotide excision repair
Large scale damage repair
What direct reverals mechanism can reverse the alkylation of guanine?
Removal of methyl group by methyltransferase -> O6 methylguanine (doesnt pair w/ C)
Methyltrasnferase recognises DNA kink + uses exposed SH to bind methyl group -. removed from DNA
How is dimerization from UV damage directly reversed?
Photoreactivation - DNA photolyase detects dimers in dark, when exposed to light it induces removal of bond between thymine dimer (pyrimidine)
-> free to base pair
not 100% effective
Summarise base excision repair
Identifies damaged base pair (lesion) using glycosylase -> hydrolyses glycosidic bond leaving abasic sugar behind.
Endo-exonuclease sutes 3’ & 5’ end removing abasic sugar + polymerase adds a new one.
Ligase connects adjacent basic sugars
Why are there many different types of glycosylase?
1 for each type of lesion:
Uracil - damaged C
8-oxoG - damaged G
3-methyl adenine - damaged A
What is a limitation of base excision repair?
Does not recognise original strand so strugglr w/ mutation that has normal base but incorrect pairing (very rare)
e.g. deamination of methylated C -> G:T pairing which is identified by TDG or Methyl-CpG Domain Protein 4 (MBD4) , converts T -> C
What does nucleotide excision repair recognise?
Recognises distortions, not lesions - caused by thiamine dimer or chemical adduct on a base
Detection leads to removal of short ss segment, then filled w/ undamaged template strand
Role of UvrAB complex in nucleotide excision repair.
UvrAB complex scans DNA + finds distortion:
- A detects then exits
B melts DNA -> single stranded bubble around distortion, also recrutits UvrC
What happens in NER after UvrC recruited?
UvrC cuts DNA upstream + downstream of distortion, recurits UvrD
UvrD (helicase II) releases single stranded garment from duplex
DNA Pol synthesises new copy + liagse joins them tohether.
Uvrs specific to E. coli but eukaryotes have similar systems - e.g. damaged DNA-binding protein (DDB) heterodimer of p120 & -48 subunits recognises specfic adducts
How is NER an example of transcription couple repair?
RNA Pol stalls when it reaches distortion
- UvrA/B recruited, NER fixes damage
allows RNA Pol to remain attached, transcription not compromised
Give 3 types of large scale damage repair
Double stranded break repair
In eukaryotes:
homologous recombination
In bacteria:
Non-homologous end joining
Translesions
What is the mechanism of double-strand break repair?
No template so uses homologous recombination of chromatids.
- ends resectioned by MRX comlex extended Exo1/DNA2 -> long 3’ ssDNA strands sticky ends in damaged DNA
- enables strand invasion so non damaged strands serve as remplate for damaged ones ( Rad51 forms a nucleoprotein filament on single-stranded DNA after end resection)
- DNA pol extends damaged strand
- Ligase joins strands -> Holiday junctions (4 stranded structure)
- Holiday junctions resolved for repaired DNA
only in eukaryotes as sister chromosome needed
Non homologous end joining (NHEJ)
Ligase glues 2 ends of of dsDNA break together
- large permanent mistakes encoded in DNA but stabilises structure
What is the function of translesion?
Allows DNA Pol to cotinue replication despite disruption/damage.
y-family polymerases recruited - incorporate new base pairs independently of parent base pairing.
- replaced by DNA Pol III when beyond damaged section
VERY high error rates - only want trasnlesion when under extreme stress
machinery expressed as part of coordinated SOS response
What is horizontal gene transfer?
Transfer of genetic material that is not ‘vertical’ - inherited. It can happen between any 2 organisms, most common in bacteria.
Examples include bacteria exchanging plasmids, organelles transferring genes to eukaryotic chromosomes, and bacteria transferring genes to plants and insects.
Who described AR plasmids in 1959?
Akiba and Ochia
AR plasmids are circular DNA that carry genes conferring antibiotic resistance.
What is an example of horizontal gene transfer between bacteria and plants?
Agrobacterium causes crown galls, transferring bacterial causative genes into plant species for disease resistance.
This process allows plants to gain useful traits from bacteria.
What gene was transferred from bacteria to the coffee borer beetle?
Gene HhMAN1
This gene allowed the beetle to digest coffee.
What ability did ferns gain through horizontal gene transfer?
Survival in dark forests
This was due to a gene transfer from hornwort, an aquatic plant.
What is the significance of Plasmodium vivax in humans?
It is a malaria-causing protozoa that can stay in the human body longer than other species.
It acquired a block of genes from humans.
What is transformation in the context of horizontal gene transfer?
Uptake of free DNA from the environment.
This can involve linear or plasmid DNA.
What type of DNA is more easily damaged, linear or circular?
Linear DNA
Linear DNA is more susceptible to damage due to open phosphate groups at the ends.
What are plasmids?
Circular DNA that requires cellular machinery to replicate.
Plasmids can vary in size and copy numbers within a cell.
What roles do naturally occurring plasmids carry genes for?
- Resistance
- Virulence
- Symbiosis
These roles are important for the survival and adaptation of bacteria.
Transformation discovery
Griffith 1970s - S. pneumoniae R & S
S is virulent + R is not (no polysaccharide capsule).
But when R strain incubated w/ heat killed S strain -> mice died
- tasnfer of DNA
competence - some bacteri more transferable than others
How is transfer of linear DNA more complicated than plasmids?
Linear DNA degraded by nuclease -> ssDNA
- ssDNA bound by competence-specific proteins that prveent its degradation
- RecA incorporates ssDNA into chromosomes
Describe conjugation as method of HGF
Bacterial ‘mating’ - requires cell cell contact
F plasmids in E.coli encode conjugation machinery
- has tra apoeron, codes for sex pilli
Pilli made by donor cell which find + attaches recipient(F- cell) + pulls it in (triggered by dircet cell contact, highly efficient)
- DNA of donor plasmid nicked on 1 strand
- Helicase spearates strands
- DNA pol synthesises other strand when it has been transferred over via conjugation pilus
How are plasmids inherited differently based upon their copy number?
Large number - inherited by random diffusion, daughter cells likely to contain plasmid due to large quantity in parent
Low number - random diffusion could skey plasmid inhertiance SO many plasmids code their own segregation machinery
can co-opt existing machinery by tying themselves to centromeres
Describe trasnduction as a method of HGF
Transfer of DNA through viral host
- disocvered using p22 bacteriphage infection of salmonella
Phage picks up host DNA + inadvertantly transfers to recipient
- can pick up host DNA upon lysis of host cell
Cannot always destroy host so transferred DNA integrated into host chromosome through lysogeny
-> host genome divides, proliferating trasnferred DNA
tRNA structure
Clover shape - has intramolecular base pairing
2 functional ends:
- anticodon
- 3’CCA end where a.acid binds, added by amino-acyl tRNA synthetase (uses ATP)
Specific tRNA bases (dihydrouridine and inosine) affect base pairing + allow more complex tRNA secpndary structure.
Ribosome structure
Has 50S (contains PTC) & 30S (mRNA channel w/ decoding sites for binding tRNAs) subunits -> 70S
3 tRNA binding sites:
- A (acceptor) binds aminoacyl-tRNA
- P (peptidyl transfer) holds tRNA carrying nascent polypeptide chain
- E (exit) where deacylated tRNA dissociates from ribosome
Making ribosomes very costly (energy) - E.coli divides every 20 mins so must double protein count in this time ~60,000 ribosomes
also has peotidyl trasnferase centre (PTC) & peptide exit tunnel
How does a ribosome know where to initiate
AUG - specifies start codon Met
Shine-Delgarno sequence (AGGAGG) found 9+/-2 bases upstream of authentic start codon in bacterial mRNAs.
Mirroed by anti-SD in 30S ribosome -> stabilises 30S-mRNA interaction
SD interaction w/ anti-SD in 16S rRNA key for initiation site selection
Function of riboswitches to control prokaryotic protein synthesis
Made of aptamer + expression platform:
ligand binding eg metal ions, amino acids or nucleotides can block or reveal access to SD/ribosome binding site by stabilising mRNA secondary structure
‘on switches’ - expression correlates w/ ligand binding e.g. YkkC binds a guanidine activating expression of genes encoding export pump
‘off switches’ - ligand binding causes ‘off’ conformation
e.g. AdoCbl riboswitches sense coenzyme B12 & switch off enzyme synthesis via feedback control to stop excess metabolite production
Initiation of prokaryote translation with IFs
IFs guide Met-tRNA binding to AUG at P site in ribosome.
- IF1 blocks A site to tRNA-met, inhibits premature 30s-50s interaction
- IF2-GTP tags tRNA + regulates entry into ribosome
- IF3 inhibits premature 30s-50s interaction, stabilises free 30s, accuracy check for tRNA-met binding
Elongation in prokaryotes
Ternary complex = aminoacyl tRNA-EFTu-GTP
- binds A site + decodes
- Peptide bond formed, peptidyl transfer of a.acid from P to A
- Association of EF-G-GTP + ejection of empty tRNA from P site
- Ribosome translocates, freeing up A site - peptidyl tRNA now in P site
EF-Tu – mediates aminoacyl-tRNA entry to ribosome
EF-G mediates translocation
Termination of prokaryotic translation
- RF-GTP binds A site where termination codon appears
- RF1 (UAG), RF2(UGA) & RF3-GTP hydrolyse polypeptide chain from tRNA
- tRNA & RF dissociate
Ribosome targeting antibiotics
Prevent translation or lter its fidelity by targeting ribosomal functional centres
30S antibiotics target decoding step in elongation (prevent it or allow errors)
- e.g. tetracycline, neonmycin, streptomycin
50S antibiotics interfere w/ peptide bond formation (via PTC)
e.g. streptogramin A/B, chloramphenicol
^bacteriostatic
Antibiotic resistance through ribosome mutations
Mutations prevent antibiotic binding
-> can give reistance to other antibitoics as there is significant overlap in binding sites
VRSA (S. aureus) caused by mutation in vanA gene, trasnposon or HGF.
How is the eukaryotic initiation phase different to prokaryotes?
- mRNAs have single ORF , 5’ cap + polyA tail vs prokaryotic mRNAs often in operons
- nuclear membrane w/ splicing
- transcription + translation separated vs prokaryotes it is coupled.
So ribosomes recruited to 5’ cap & then scan to locate Kozak sequence + AUG start codon
5’ & 3’ UTRs vital for translation control
Scanning mechanism of initiation in eukaryotes
Initiator tRNA binds 40S subunit, ribosome binds near 5’ cap, but in wrong place.
43S PIC binds mRNA -> 48S, ribosome moves along sequence scanning to find AUG (1st one found is used unless it has poor kozak sequence)
Large 60S subunit joins
How is the 43S pre-initiation complex formed in eukaryotic initiation?
- disassembled 40S subunit needs to bind eIF1, 1A & 3
- eIF2 binds met-tRNAi when bound to GTP -> ternary complex (TC)
- both combine w/ eIF5 to form 43S PIC
Initiator met-tRNAi binds 40S ribosome in complex w/ eIF2 (+1, 1A, 3 & 5)
How is mRNA selected + 48S PIC formed in eukaryotic initiation?
mRNAs primed by eIF4E binding at 5’ cap w/ eIF4G & poly-A binding protein (PABp)
-> mRNAs circularise forming closed loop complex
eIF4A/B unwinds any mRNA secondary structure -> landing site for 43S complex near 5’ end
43S lands so met-tRNAi & mRNA can base pair - codon/anitcodon interaction can be foun during scanning
Factor release & 60S binding in eukaryotic initiation
AUG codon-anticodon pairing triggers eIF1 release + eIF2-GTP hydrolysis (does not bind met-tRNAi so released)
eIF5B-GTP binds, promoting 60S association + other factors released
Eukaryotic elongation factors
eEF1A-GTP: binds all tRNAs except initiator to A site (EF-Tu), activated by eEF1B (GDP/GTP exchanger, EF-Ts)
eEF2-GTP: mediates translocation (EF-G)
eEF5A: assists in peotide bond formation (EF-P)
Eukaryotic termination factors
eRF1: recognises all 3 stop codons in A site + catalyses release of nascent polypeptide from tRNA in P site
eRF3-GTP: regulates ribosome binding via GTP hydrolysis
Why are global translational controls needed?
- increases speed of effect on protein levels
- allows local control in specific part of cell
Translation often inhibited transiently while stress is resolved.
Can be activated -> faster growth, upon stimulation by hormones, GFs, mitogens + cytokines
- globally, during development
- locally, wound healing
5’ cap & 3’ polyA tail on euakryotic translation
- synergistically enhance translation of all eukarytic mRNAs
Conducted experiment w/ mutants of both elements- luciferase used as reporter gene (fluorescence).
- found to be vital for translation, RLU decreased in mutants.
bind eIFs to direct ribosome entry & binding
How can global protein synthesis activity be measured?
Polysome profiling - inidcates levels of 40S, 60S, 80s + polyribosomes.
Cells treated with cycloheximide to freeze all ribosomes at current positions on mRNA.
- cell extracts prepared + centrifuged
Can compare under differetn conditions.
e.g. increasing H2O2 inhibits protein synthesis due to oxidative stress
- new proteins can also be labelled w/ 35^S methionine
Global translation control by phosphorylation of eIF2
Phosphorylation of eIF2 at a-serine 51by Gcn2 inhibits eIF2B GEF activity.
-> translation attenuated as ternary complex not formed
Transient stress repsonse is good, but prolonged is bad (see footnote)
aberrantly high eIF2A phosphorylation by PKR kinase -> seen in brain w/ Down Syndrome, protein synthesis needed for LTP + synaptic plasticity (associated w/ memory)
Function of 4E-BPs as a global translational control
eIF4E binding proteins inhibit translation
- prevent 4E intract w/ 4G
- mRNA recuritment blocked so translation shut down
4E-BPs & eIF4g have common sequence motif
-> YXXXXL(delta), binds same part of eIF4E
Control mechanism of 4E-BPs
mTOR (kinase) promotes growth & translation by inactivating 4E-BPs via phosphorylation.
5’TOP mRNAs that have 5’ terminal oligopyrimidine run (C/U) are most affected by 4E-BP1/2 mTOR control.
- found in ribosomal proteins + TF mRNAs, so control tranlation of everything else
Thr36 + 45 are key phos sites -> dramatic folding from a-helix to B-strand
- new folding buries 4E-BP Y53 in B-strand so lowers affinity for eIF4E >1000 fold
eIF4E free for translation
5’TOP - 5’ terminal oligopyrimidine
adding 4E-BP1 slows translation, slowing tumour growth in breast cancer cells
Specific mRNA controls of initiation
Work w/ global controls -> precise regulation of transaltion specific proteins.
Features for controls:
- 5’cap & polyA tail promote translation on all mRNAs
- 5’ & 3’ UTR critical for controlling translation
1) Ferritin mRNA
2) Arg-2 mRNA
3) Nanos mRNA
Ferritin mRNA initiation control
Iron levels in mammals - aconitase/IRP1 regulates ferritin mRNA translation.
High [Fe] - aconitase is a 4Fe-4S cluster binding enzyme, converts citrate -> isocitrate in TCA cycle
- RNA helicase (eIF4A) allows 40S scan to find ORF AUG
Low [Fe] - iron limiting, aconitase inactivated, transforms into IRP1 -> binds stem loop structure (IRE) in ferritin mRNA 5’UTR
- prevents 40S access + scanning
Arg-2 mRNa initiation control
Arg levels in neurospora. R biosynthesis regulated by both R levels & a uORF encoding AAP
- Arg-2 makes R when R levels low, repressed by high R
Control requires:
- normal scanning
- leaky scanning of AAP uORF to translate Arg-2
- ribosome & AAP sequence to monitor excess Arg
Low Arg - leaky scanning of uORF (only 50% ribosomes translate AAP, poor kozak sequence), so ARG-2 translated, those that translate AAP dissociate from mRNA
High Arg - excess Arg stalls ribosomes translating AAP at stop codon, Arg binds AAP peptide in exit tunnel blocking AAP release
- stalled ribosomes block further ribosome movement
- Arg-2 not translated
AAP - Arg attenuator peptide
Principle: uORF in 5’UTR can control ribosome access to a main ORF + bring about condition-specific regulation, ~50% human mRNAs have uORFs.
Nanos mRNA initiation control
Early embryonic development in fruit fly. A-P axis must be established - requires post-transcriptional control of maternal mRNAs.
Cup protein is 4E-BP that regulates translation + location of Nanos protein.
Nanos repressed everywhere except posterior end - directed by mRNA 3’UTR binding proteins (Smaug & Glo)
- interact w/ stem loop RNa structure in 3’UTR
- form closed loop w/ Cup so eIF4G cannot access mRNA
- ensures only specifc mRNas transaltionally repressed -> localisation in a cell + polarity
Nanos is a translational regulator of hunchback
Principle: 4E-BP tethered to sequence or structure in 3’UTR via specific RNA-binding proteins can control ribosome access -> spatially regulated translation
- Also happens in mammals inc. neurons to regulate memory
Specific mRNA control of elongation
Antizyme mRNA - promotes programmed frameshifting in mammalian cells
Common in viruses (HIV, SARS-Cov2) - antigenic drift.
In eukaryotes: +1 frameshift controls polyamine levels in cells (spermine, spermidine, putrescine, ornithine)
AZ levels controls ODC levels -> tight control of polyamine levels.
- delivers ODC to proteasome for degradation + inhibits polyamine uptake
mRNa has 0 frame (ORF1) w/ stop codon + +1 frame w/ no start codon
SO ribosome translates ORF1 OR longer ORF1-ORF2 (which is functional antizyme enzyme)
3’ pseudoknot is modified stem loop, stalls ribosome so it can attach 2nd reading frame (has shifty segment)
ODC - ornithine decarboxylase enzyme
Excess spermidine promotes ribosome frameshifting feed back control by binding ribosome.
Principle: elongation + reading frame maintenance can be affected by combination of local sequences + small molecules binding to ribosome.
Processing of RNA Pol II derived RNAs
- 5’ end capping (GTP binds 5’ end + 2’ pos of 1 st 2 nucleotides methylated)
- intron splicing: signalled by conserved sequences + branchpoint region (spliceosome is complex of snRNPs)
-> U2 + U5 + U6 make up active site - 3’ end cleavage + polyadenylation (CPSF binds AAUAA, CstF binds G/U -> Poly-A pOlymerase + cleavage factors recruited)
+ mRNA specific events: RNA editing, alternative splicing
mRNA cap structure
7-methylguanosine connected to 5’ end via triphosphate bridge.
Recognised in nucleus by CBP20/CBP80 -> stability, connection to export apparatus + connection to splicing (lariat formation)
In cytoplasm: interacts w/ eIF4E -> RNA stability, nuclear export + translation initiaiton
its location dictated by site of transcription - regulated by alterantive promoters
mRNA splicing
1993 Roberts + Sharp shared Nobel prize - hybridized denatured adenoviral DNA fragment to purified exon mRNA
Conserved consensu sequences define splice site.
e.g. Branchpoint (YNYURAC) is 30 bases upstream of 3’ end - connected by polypyrimidine tract
1) 5’ splice site binds to 2’-OH at branchpoint forming loop (spliced from exon 1)
2) exon 1 joins to exon 2 via 3’ splice site -> phosphodester linkage (upstream introns spliced out)
U1, 2, 4, 5 + 6 snRNAs very important
- U1 frees 5’ end so it can base pair to specific sequences -> snRNPs precisely target intron/exon boundaries
Y - pyrimidine (C & U)
R - purine (A & G)
N - any
Splicing accessory factors
- Commitment/E complex formed by SR-U2AF interaction
- U2 then recruited
SR associates w/ RNA Pol II tethering transcription to splicing
What is alternative splicing?
Exons of primary transcript reconnected in various ways -> altered protein products (isoforms)
- relies on efficieny of splice signals (donor/acceptor)
- extra enhancer/silencer sequences control this efficiency
SR proteins bind splicing enhancers dictating where it happens
hnRNP protein (hnRNPA1) binds silencing enhancers
many different effects on protein
Examples of alternative splicing in flies
Dscam1 mRNA processing
Every neuron in fruit-fly expresses different version on its surface.
- tiny variations in specificity + function between isoforms
-> 38,016 different mRNAs from 1 primary transcript
What are the main pathways of mRNA decay?
5’ -> 3’: deadenylation , decapping & 5’->3’ exonuclease activity
3’ -> 5’ exosome: deadenylated mRNas degraded by exosome
^ redundancy in both pathways- can pick up each other slack
Describe the 5’ -> 3’ degradation pathway
- pop2/Ccr4 complex -> deadenylation
- Lsm1-7 binds polyA after PABP removal + recruits decapping proteins
- Dcp1/2 carry out decapping of 5’ end.
- Xrn1 nuclease cleaves mRNA
PABP - polyA binding proteins
Lsm1-7 is a marker protein complex -> necessary to recruit decapping proteins.
Describe the exosome (3’->5’) mRNA degradation pathway
- pop2/Ccr4 complex -> deadenylation
- Exosome/Ski complex binds 3’ end -> 9 subunits w/ ring shape
-> has 3’->5’ exonucleases + other RNases, RNA helicases, porcess other RNAs - Dcp1/2 removes 5’ end left over (scavenger decapping enzyme)
What are P bodies?
Cytoplasmic bodies that contain:
- components to 5’->3’ decay pathway
- components to RNAi pathways
Possibly store mRNA for future degradation or use.
mRNA scrapyard
How is mRNA surveilled & marked for degradation?
Cap binding complex inhibits acces of deadenylase + decapping enzymes.
- inhibition of initiation will promote mRNA decay
Aberrant mRNAs are degraded via specialised mRNA surveillance pathways:
- NMD
- NSD
- NGD
Nonsense mediated decay (NMD)
Identifies mRNAs w/ premature stop codon.
- either due to mutation/errors or incomplete splicing
EJC & CBP20/80 recruits 3 UBF proteins to where ribosome stalled
-> UBF1 phosphorylated & mRNA endonucleolytically cleaved
Exon junction complex (EJC) is a marker for where splicing has occurred
Non-stop decay (NSD)
Identifies mRNA with no STOP codons
- either stop codon mutated or premature polyadenylation occurs
Lack of stop codon means ribosome displaces PABP (acts as buffer) - UPF recruited, then exosome for 3’->5’ decay
No-go decay (NGD)
Deals with issues during translation.
If ribosome stalled due to secondary structure - causes decay.
Dom34/Hbs1 complex recruited to stalled ribosome - dissociates ribosome + prootes endonuclease cleavage.
5’ end mainly degraded by 3’->5’ but both pathways used (redundancy)
How can mRNA decay be controlled by cis acting elements in the UTRs and ORF of mRNA?
Regulates amount of mRNA made.
3’UTR examples include:
1) regulation of mRNA stability by AREs
2) Transferrin receptor mRNA & IRP1 (IREBP1)
ARE - AU rich element
Regulation of mRNA stability by AU rich elements
i- usually destabilisng but can be bound by proteins which stabilise it (impacts onTFs, cell cycle, cytokine/lymphokines)
-> proteins include: AUF1 (destabilisation of c-fos, c-myc, IL3) , HuR (stabilises AU elements in Cyclin A1/D1) & TTP
Destabilisation - RBP recruited + interacts w/ decapping proteins + deadenylation complex
Regulation of mRNA stability by transferrin receptor mRNA & IRP1 (IREBP1)
- IRP1 binds stem loops in Tfr 3’UTR + blocks access of ARE-BPs -> stabilising mRNA
- Fe binding to IRP1 causes its dissociation from AU rich sequences -> ARE-BPs can access mRNA + induce degradation
self regulating mechanism - swithes off when plent of Fe in cell
What is RNAi and its mechanism?
Post-transcriptional gene silecnign mediated by short RNA mols i.e. miRNA/siRNA
Initiation step - siRNAs generated
Effector step - degradation of target mRNA
Describe the initition step of RNAi
Microprocessor converts pre-miRNA into ds-stem loop structure.
- dsRNA cleaved by dicer + phosphorylated by putative kinase
- RdRp amplifies siRNA (not in mammals)
Pathway above generalised - actually species specific
What is the dicer protein?
Atp-dependent helicase w/ dsRNA binding domain & RNase III activity.
- only 1 in C. elegans/mammals but > 1 in Arabidopsis/Drosophila
Work as dimers to generate small 22-24 bp siRNAs
Describe effector step of RNAi
siRNA binds mRNA by base pairing + unwinds
RISC activated to:
- inhibit translation in animals
- degrade mRNa/ silence heterochromatin in plants
RISC - RNA-induced silencing complex
Structure of RISC
- 2 RNA binding proteins
- RNA/DNA helicase
- translation initiation factor
- RdRp in some species where effct needs to be amplified
- TM protein -> propagates initiation across cell pop.
RISC-siRNA -> mRNA degradation
miRNP-miRNA -> tranlsation initiation
RITS-rasiRNA -> heterochromatin modification
All have argonaute proteins (indicative)
Non-coding RNA
RNA that doen not encode protein but may stoll contain info or have function.
Encoded by RNA Pol II:
short - snRNA, snoRNA, miRNAs, siRNAs
long - lincRNAs, eRNAs, PROMPTs
rRNA & tRNA encoded by RNA Pol I & III
Depednently synthesised ncRNAs originating from nucleosome free regions (NFRs)
Dependent so co-expressed w/ mRNA
Inc promoter associated (paRNA) or transcription start site (TSS-ncRNA).
Transcribed in same direction as sense mRNA, 20-60nt long + non-functional
- BUT important check in point in gene expression regulation
How are PROMPTs generated?
Trabscribed in revrse orientation to sense mRNA -eukaryotic promoters often bidirectional so trancription can initiated both directions.
0.5-1.5kb long, non-functional
If ncRNAs not terminated - transcription would evetually be blocked by Pol transcribing PROMPTs
- uncontrolled pervasive transcription -> lethal interference
Pol II blocking each other -> tension -> DNA breaks
How is pervasive trasncription between paRNA/TSSRNAs & PROMPTs avoided?
Integrators + restrictors used.
Transcription of ncRNAs can be initiated anywhere inc terminators
Non-productive veents immediately restricted to avoid interference
Dependent ncRNAs processed from pre-mRNA intermediates
small nucleolar RNAs (snoRNAs) are embedded in introns.
Co-assmebled during mRNA splicing - trimming takes place to form mature snoRNP from an intron lariat.
- uses 5’ and 3’ degradation
Functions:
- RNA methylation (box C/D)
- RNA pseudouridinylation (box H/ACA)
they recognise RNA targets via short complementary sequences
mainly independent in lower hosts
snoRNas in human disorders
Prader-Willi caused by large DEL in chrom 15 - snoRNa cluster involved, some can form long non-coding RNAs (sno-lncRNAs)
Requires 2 sno-RNA in same intron SO exonuclease cannot degrade -> very stable mol w/ sno-RNA at both ends.
Sno-lncRNAs one of most abundant RNA species in human stem cells
Independntly expressed ncRNAs
Expressed independently of protein-coding genes - transcribed from own promoters
small nuclear RNAs (snRNAs)
- in all eukaryotes (pre-mRNA splicing)
- for riboprotein complexes (U1, 2, 4, 5, 6)
- short + fold into structural RNAs
- terminated by integrator as non-polyadenylated
^ all transcribed by Pol II except U6 (Pol III)
Enahncer RNAs (eRNAs) also independent
- cis-regulatory elements, cooperate w/ promoters to regulate expression
- highy transcribed
- terminates by either cleavage & polyadenylation , restrictor/integrators complexes
eRNA structure & function
Non-polyadenylated - bidirectionally transcribed so terminated by restrictor/integrator complexes
Polyadenylated - unidirectionally transcribed (>4kb), trasncirbed by v. active enhancers, terminated by cleave + p. adenylation
Regulation of expression (putative):
- molecular glue
- sequestering
- phase separated environemnt
Long intergenic ncRNAs
lincRNAs - independent transcription units
- many function + tissue specific
BUT synthesis more inefficient than mRNA + mainly in nucleus
Mostly associated w/ higher order structure + binding proteins.
- associated w/ many different diseases
lincRNAs are ‘non-coding copies’ of mRNAs: capped, spliced & polyadenylated. lincRNAs usually localised in the nucleus + many lincRNAs are functional
Transposable elements
DNA sequences that have ability to change position within a genome.
Either replicate sequence + insert into another gene (copy + paste)
OR sequences removed from one locus + moved to another (cut + paste)
46% human genome
History of TE discovery
Barbara McClintock studied mechanism of unstable inheritance of mosaic colour patterns in maize seeds.
Described loci disscoiation (Ds) & activator (Ac) - both could change genome.
Progenitors of TEs likely present in common ancestor of all eukaryotes
- originates from viruses or actively transcribed small RNA genes
Class 2 TEs
DNA transposons - mobilised via DNa intermediate
- cut & paste mechanism
- flanking direct repeats play role in TE insertions (left behind as footprints after TE excised)
Inverted terminal repeats (ITRs) complementary to each + recognise transposase
Class 1 TEs
Retrotransposons rely on RNA Pol & reverse transcriptase
- RNA interemdiates & copy-paste mechanisms
Subclasses are long terminal repeats (LTRs) & non-LTRs (LINE & SINE)
LTR subclass of retrotransposons
Presence of LTRs, produces target site duplications (TSDs) upon insertion.
ORF encodes rev. transcriptase, ribonuclease H + integrase -> enzymatic machinery for integration
LINEs
long interspersed elements:
- pol II promoter
- ORF1 encodes RNA binding chaperone
- ORF2 encodes endonuclease -> ss nick in DNA, rev. trasncriptase uses nicked DNA to prime rev. transcription
- polyadenylation site followed by A(n)
non-LTR subclass
SINEs
short interspersed elements:
- do not encode any of own retrotranscription machinery, mobilised by L1 machinery
- contains components of RNA Pol III promoter
- Alu SINEs contain Alu restriction site
Why are TEs not ramdomly distributed in the genome?
- If strongly deleterious, rapidly removed
- If little/no effect then any reach fixation
rarely fixed in exons & depleted from regions coding ncRNAs
How does transposition contribute to genomic instability?
Non allelic homologous recombination (NAHR)
e.g. MLL-1 & BRCA1 hotspots fror Alu-Alu recombination
-> gene duplications, rearrangements + deletions
BRCA genes TE insertion -> breast cancer
How to TEs drive evolution?
Especially in eukaryotes - limited HGT like in bacteria.
- Can shuffle exons or even become them.
- Faciltate translocation of genomic sequences
e.g.
- Rag1 & Rag2 catalyse sonatic recombination in vertebrates
- Gag-derived Arc gene involved in memory + synaptic plasticity
Gag polyprotein integral to viral structures
How can TEs be repressed?
Human cells employ chromatin remodelling.
- HUSH & KRAB-ZNF regulates methylation of Lys9 (H3K9me3)
- Polycomb complex mediates H3K27me3
-> heterochromatin formation to keep TEs inactive
Organelle genomes
Small + compact, genes relocated to nucleus -> better control mechanisms
Peptide sequences have targeting signal -> directs proteins back to organelle
mtDNA
- maternally inherited
- different structure + code to nuclear genome
Bidirectional replication by DNA Pol gamma, leading strand synthesis starts at specific open D-loop
Human mtDNA is circular loop - transcribed from both strands as long polycistronic precursor
- pre-mRNAs processed by tRNA punctuation model
tRNA punctuation model for mtRNA
tRNAs act as punctuation marks for mtRNAs -> excised by endonucleases (RNase P/Zs, Z cleaves 3’ end, P cleaves 5’ end)
22 interspersed tRNAs removed to release rRNAs + mRNas
In higher eukaryotes, RNase P encoded in nucleus then imported into mt
Gene expression in mitochondria
Has its own ribosomes.
In mt mRNA:
- no cap
- no to be spliced
- polyadenylated after transcription by mtPAP
shorter polyA tail in mtRNA
PNPase function
In mt, chloroplasts + bacteria, have exonucleolytic + polyadenylation activity.
Human PNPase is exonuclease only
-> degrades RNA
ATP dependent hSUV3 unwinds secondary structures
Examples of mitchondrial disorders
MELAS
MERRF
LHON
mtDNA mutation rate ~30 fold higher than
mtDNA heteroplasmy
Same cell can tolerate varying proportions of mutated & normal mtDNA
Therapies to remove mutated mtDNA
Heteroplasmy shifting - targeted removal of mutated mtDNA
OLD: mitochondrially targeted restriction endonucleases
NEW: programmable nucleases w/ sequences specific DNA binding domains:
- zinc finger nucleases (ZFNs)
- transcription activator like effector nucleases (TALENS)
^ linked to seuqnece independent endonuclease (Fokl)
cpDNA
- maternally inherited, codes for proteins in photoysnthesis
- has same genetic code as nuclear genome
2 origins from which elongation of nascent strands initiated - fork movements towards each other + fusion of D loops (bidirectional)
highly conserved genome, excised by exo- and endonucleases
cp mRNA
- no cap
- may have introns
- polyadenylation marks for degradation
