Pro & Euk Flashcards
Prokaryotic vs Eukaryotic Genome
- Size + Appearance
- Prokaryotes have smaller, singular circular genome w fewer genes
- Eukaryotes have larger, multiple linear molecules w more genes - Association with proteins
- Yes, relatively fewer histone-like proteins for prokaryotes, histones and scaffold proteins for eukaryotes - Location
- Prokaryotes: Nucleoid region + plasmids
- Eukaryotes: Membrane bound nucleus + no plasmids - Non-Coding Sequences
- Prokaryotes typically have less than 15% of genome as ncDNA, few repeated sequences
- Eukaryotes have 98% DNA as ncDNA, many repeated sequences - Introns, Enhancers/Silencers
- Prokaryotes have no introns, few enhancers/silencers
- Eukaryotes have many of both, for almost every exon - Operon
- Prokaryotes have many, eukaryotes very rare, each gene has its own promoter - Ori
- Prokaryotes only have one, replication ends at terminus region
- Eukaryotes have many, replication ends at telomeres - Level of coiling
- Purpose is to compact DNA for nucleus, prevent DNA damage and regulate gene expression and transcription
8a) Prokaryotes: Relatively low, some looping around his tone like proteins
- Unfolded chromosome diameter is 430 um
- DNA folded into chromosomal looped domains by protein-DNA associations
- Supercoiling causes further compacting, final chromosome is around 1 um
8b) Eukaryotes: High and complex packing
- DNA double helix is negatively charged while histones are positively charged, thus held together by electrostatic interactions
- Most DNA wound around octamers of 8 histones to form nucleosomes, forming a 10nm fibre. Linker DNA joins adjacent nucleosomes
- 10nm fibre coils around itself to form 30nm solenoid
- Solenoid forms looped domains when associated with scaffold proteins
- Supercoiling produces metaphase chromosome
Introns
- Found between exons with no involvement in translation and are excised during splicing to join exons in mature mRNA
- Spliceosome recognises GG at 5’ end and AG at 3’ end to fold into a loop and excise
Promoters
- Control elements just upstream of transcription site
- TATA box at -25 sequence serves as recognition site for binding of GTFs and RNA, determining precise location to initiate transcription
- CAAT and GC boxes improve efficiency of transcription initiation complex formation
- The more critical elements, the greater the binding efficiency and transcription frequency
Telomeres
- Non-coding tandem repeat sequences TTAGGG in humans found at end of linear chromosomes
- Single stranded region known as 3’ overhang without complementary strand
Function 1 of Telomeres
Telomeres prevent loss of vital genetic information due to the end-replication problem
- The end-replication problem occurs because DNA pol requires a free 3’ OH end on pre-existing strand to add free nucleotides
- However, on lagging strand, RNA primer is removed without replacement, creating a 3’ overhang w/o complementary nucleotides, with ends shortening after every round of DNA replication
- Shortening of chromosomal ends leads to shortening of telomeres instead of genes coding for vital information, since telomeres are non-coding
- When telomeres reach critically short lengths (Hayflick limit), apoptosis is triggered
- Note: it does NOT prevent shortening of chromosomes or prevent end replication problem, it’s just like a bandaid to an inevitable problem
Function 2 of telomeres
Telomeres protect and stabilise terminal ends of chromosomes
- Telomeres form a displacement loop with 3’ overhang, preventing fusion with other chromosomes and preventing DNA repair machinery from recognising it as broken DNA and prevent it from triggering cell cycle arrest and apoptosis
- 3’ overhang linearly is similar to DNA damage
- Single-stranded DNA can also cause annealing to complementary regions on different chromosomes (problem)
Function 3 of telomeres
Telomeres allow own extension, providing an attachment point for telomerase
- Telomerase maintains telomere length in germ cells, cancer cells and embryonic stem cells, avoiding senescence (Age expectancy is thus highly genetic, bcos original telomere length is inherited from germ cells)
1. A short 5-nucleotide segment of telomerase RNA binds to specific tandem DNA repeat TTAGGG via CBP, aligning telomerase reverse transcriptase wrt DNA. Telomerase has active site complementary in shape and charge to TTAGGG
2. The adjacent 6 RNA nucleotides used as template for telomerase reverse transcriptase to form complementary DNA sequence thru CBP by catalysing formation of phosphodiester bonds
3. Telomerase moves 6 nucleotides in 5’ - 3’ direction to produce tandem repeats of TTAGGG. Strand with 3’ overhang is extended and complete
4. Using extended 3’ DNA overhang as template, primase synthesised RNA primer, with DNA polymerase adding nucleotides to 3’ OH end to synthesise a complementary DNA strand. The nick is sealed by DNA ligase and RNA primer removed
- Some parts of telomerase RNA are double stranded (achieved by CBP) to stabilise molecule. It folds into a precise 3D conformation complementary to active site of enzyme
Centromeres
- Constricted regions of non-coding DNA consisting of tandem repeat sequences on chromosomes
- Allow for proper nuclear division by:
1. Allowing sister chromatids to adhere to each other
2. Allowing kinetochore proteins and spindle fibres to attach for homologous chromosomes to separate to opposite poles - Without centrosomes, improper alignment and segregation can result in non-disjunction
Genomic Level Regulation
Main thing: RNA pol and GTFs can/cannot bind to promoter
1. Chromatin Remodelling Complex
- Activating complexes decondense DNA to make it less tightly bound to histones and allow for more access of RNA pol and GTFs to promoter
- Deactivating complexes condense DNA to make it more tightly bound to histones and allow for less access of RNA pol and GTFs to promoter
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DNA Methylation
- Addition of methyl group to selected cytosine nucleotides located in CG sequences by enzyme DNA methyltransferase
- Reduces accessibility of promoter by blocking binding of RNA pol and GTFs to promoter, preventing TIC assembly
- Also condenses chromatin by recruiting DNA-binding proteins like repressors, histone deacetylases, repressive CRCs
- Often occurs during differentiation as genome is too large, thus packaging unneeded portions into heterochromatin and prevent it from being expressed
- DNA methyltransferase inhibitors can cause demethylation, which increases expression -
Histone acetylation/deacetylation
- Acetylation adds acetyl groups to lysine residues of histones catalysed by histone acetyltransferase
- This increases accessibility by decreasing electrostatic interactions between DNA and histone by removing + charges, loosening chromatin binding and making promoter region more accessible to GTFs and RNA pol, allowing formation of TIC
- Deacetylation removes acetyl group, catalysed by histone deacetylase and restores positive charges to make promoter less accessible
Transcriptional
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Enhancers (distal control element)
- Enhancers, when bound with specific transcription factors called activators promote assembly of transcription initiation complex at promoter to increase transcription frequency
- Also recruit histone acetyltransferase and chromatin remodelling complexes to decondense chromatin and increase accessibility of promoter to GTFs and RNA pol -
Silencers (distal control element)
- Silencers, when bound with specific transcription factors called repressors inhibit assembly of transcription initiation complex at promoter to decrease transcription frequency
- Also recruit histone deacetylase and repressive chromatin remodelling complexes to condense chromatin and decrease accessibility of promoter to GTFs and RNA pol -
TATA, GC and CAAT boxes (proximal control elements)
- Located within promoter to improve efficiency of promoter by recruiting GTFs and RNA pol to promoter
Post-Transcriptional Level
-
Addition of 7-methylguanosine cap to 5’ end at pre-mRNA
- 5’ cap helps cell to recognise mRNA for further processing and export out of nucleus
- Increases half-life to prevent digestion by exonucleases
- Promotes translation initiation as 5’ cap recognised by eukaryotic initiation factors - **Alternative RNA splicing of a single pre-mRNA produces mature mRNA with different combinations of exons, producing different protein isoforms
- Non-coding intron sequences excised and exons joined to form mature mRNA
- Points of excision carried out precisely by spliceosome (snRNA-protein complex), thus 1 gene codes for > 1 type of polypeptide -
Addition of 3’ poly-A tail
- 3’ end cleaved enzymatically downstream of highly conserved polyadenylation signal (AAUAAA) by endonuclease
- Immediately after, poly-A polymerase adds series of AMPs to form poly-A tail
- Enhances half-life of mRNA transcript by preventing digestion by exonucleases
Translational Level
- Translation is initiated when small ribosomal subunit, eukaryotic initiation factors (eIFs) and initiator tRNA form a complex which binds to 5’ cap and poly-A tail, causing mRNA to circular rise. Complex locates AUG start codon and binds large ribosomal subunit to complete translational initiation complex
1. mRNA stability/half-life -
Longer 3’ poly-A tail = more stable mRNA w longer half life which can serve as template for translation longer, more polypeptides translated
2. Anti-sense RNAs -
CBPs to part of mRNA to form double stranded RNA, blocking translation and targeting it for degradation by ribonucleases
3. Binding of translation repressors - Bind to 5’ cap, 5’ UTR or 3’ UTR to prevent assembly of translation initiation complex
4. Activated translational initiation factors needed to form TIC
Post-translational Level
-
Covalent Modifications to form functional proteins
- Glycosylation, disulfide bond formation, attachment of prosthetic groups - Proteolytic Cleavage to make functional enzymes (often synthesised inactive form)
-
Phosphorylation/dephosphorylation to regulate protein activity
- By kinases/phosphatases to make enzyme active/inactive eg in phosphorylation cascade -
Protein degradation
- Ubiquitin ligase catalyses addition of ubiquitin to unneeded protein
- Ubiquitin-tagged protein has peptide bonds hydrolysed by proteasome, cleaving it into smaller peptides by sequestering it within central cavity
- Proteasome and ubiquitin recycled and peptide further degraded
In prokaryotes
- Operons (inducible/repressible, refer to Bacteria)
- mRNA stability
- prokaryotic mRNAs have a relatively short half life because they do not undergo post-transcriptional modification
- This allows bacteria to rapidly adjust protein synthesis in response to environmental changes by controlling gene expression
Cancer Causative Factors
- Heredity
- Gene mutations in germ cells/epigenetic patterns passed down from parents
- eg BRCA1 gene is a TSG responsible for DNA repair, loss of function mutations of which severely increase risk of breast and ovarian cancer -
Chemical Carcinogens
- Most carcinogens covalently bind to DNA to form adducts that cause miscoding, which cause such massive DNA damage it overwhelms repair enzymes
- Substances such as benzene, benzopyrene, formaldehyde, phenols and nicotine in cigarette smoke -
Ionising radiation
- UV light directly damages DNA by breaking H bond between nucleotides to form dimers - Loss of immunity from viral infections
- HIV infections weaken immune system and reduce ability to fight tumours that might lead to cancer —> several 1000x more likely to cause Kaposi’s sarcoma, 70x more likely to get non-Hodgkin’s lymphoma
- Human PPV infection produces E6 protein that destroys p53 protein, increasing risk for cervical cancer
