Prokaryotic & Eukaryotic Genome Flashcards
Compare eukaryotic and prokaryotic cells
Size Nucleus Organelles Ribosomes Cell wall Plasma membrane (S)
Compare the prokaryotic and eukaryotic genomes (exclude organisation)
Size & no. of genes Appearance Molecule Association w proteins Location Extrachromosomal DNA Level of DNA packing/coiling
Why is the packing of DNA important?
- compact–> fit in nucleus (eu)
- regulate gene expression (eu)
- prevent DNA entanglement and breakage
Describe how DNA is packed in eukaryotes
- -vely charged DNA double helix winds arnd octamers of 8 +vely charged histone proteins via electrostatic interactions. → nucleosome (10nm fibre). Linker DNA joins adjacent nucleosomes.
- 10nm fibre coils around itself→ 30nm chromatin fiber/solenoid (interphase DNA)
- 30nm fibre form looped domains when associated w scaffold proteins→ 300nm fibre
- Supercoiling→ metaphase chromosome
Describe how DNA is packed in prokaryotes
DNA double helix folds into looped domains by protein-DNA associations→ supercoiling w the help of DNA gyrase & topoisomerase
Compare the organisation of eukaryotic and prokaryotic genome
Non-coding regions - introns - promoters (s) - enhancers/silencers - repeated seq Operon Ori
Explain why the end replication problem occurs
DNA polymerase needs free 3’ OH grp to add free nt to growing DNA strand→ RNA primer synthesised to provide the free 3’ OH end for the addition of nt
⇒ 5’ end of newly synthesised strand will have RNA primer removed wout replacement of DNA due to no 3’ end available for DNA polymerase→ 3’ overhang → chromosomes shorten every replication ⇒ shortening of telomeres
Describe the role of telomeres
Definition: non-coding DNA made up of a series of tandem repeat seq, found at both ends of eu chromosomes
- Non-coding telomeres ensure vital genetic information are not lost due to end replication problem, where daughter chromosomes shorten at the telomeres w each round of DNA replication, as DNA polymerase can’t replace RNA primers w DNA
- Telomeres protect and stabilise terminal ends of chromosomes, by forming a loop w 3’ overhang, preventing ends of chromosome from fusing w those of other chromosomes→ prevent DNA repair machinery from recognising ends of chromosomes as DNA damage, protecting the chromosome, prevent apoptosis
- Telomeres allow for own extension, as 3’ overhang providing an attachment point for the correct positioning of telomerase enzyme
Describe the function of telomerase
- AS complementary in conformation and charge to a specific telomeric DNA sequence
- Catalyses the formation of phosphodiester bonds
translocate in 5’ to 3’ direction→ series of tandem repeats of GGTTAG→ elongate telomere - Telomerase RNA
> Anneal via cbp w ss 3’ overhang of telomere→ align telomerase in correct orientation wrt DNA
> Template for elongation of 3’ overhang, synthesise complementary DNA seq via cbp, where A pairs w U, T w A, C w G→ tandem repeat seq
Describe the role of centromeres
Structure: seq of non-coding DNA region of chromosome made up of tandem repeats
- Allowing sister chromatids to adhere to each other
- Allow kinetochore proteins & subsequently kinetochore microtubules to attach→ bivalents can be aligned along metaphase plate during MI of meiosis + chromosomes aligned singly along metaphase plate during M/MII of meiosis→ kinetochore microtubules shorten and separate them to opposite poles during A/AI/AII
⇒ allows proper alignment and segregation of chromosomes
Describe splicing
introns excised, exons joined tgt by spliceosome, a snRNA (small nuclear RNA)-protein complex, which recognises seq at intron-exon boundary→ produce functional proteins
Describe alternative RNA splicing
different exons of a single pre-mRNA joined to produce different mature mRNAs → one gene can code for >1 polypeptides; diff proteins can be produced
What is the function of promoters?
Recognition & binding site for GTFs, which recruits RNA polymerase→ transcription initiation complex→ initiate transcription
- Critical elements: TATA box determines precise location of transcription start site
What is the function of enhancers ?
- positive regulatory elements
- Recognition & binding site for specific transcription factors called activators→ promote assembly of transcription initiation complex by…
→ bending spacer DNA allows activators to bind & stabilise transcription initiation complex at promoter
→ recruit histone acetyl transferase & crc to decondense chromatin, ↑ accessibility of RNA pol & GTFs to promoter
⇒ upregulate/↑ freq of transcription; gene activation
What is the function of silencers?
- Negative regulatory elements
- Recognition & binding site for specific transcription factors called repressors→ prevent assembly of transcription initiation complex by…
→ interfere w action of activator by: competitive DNA binding, masking activation surface & bending spacer DNA for repressor to directly interact/bind to GTFs
→ recruit histone deacetylase & repressible crc to condense chromatin, ↓ accessibility of RNA pol & GTFs to promoter
⇒ downregulation/ ↓ freq of transcription; gene silencing/repression
Explain the importance of gene regulation
- Cellular differentiation: Regulation of gene expression→ each specialised cell hv different genes expressed→ different, tissue-specific proteins synthesised→ diff function
- Adapt to changes: proteins produced depends on circumstances & demand
- Conserve resources: transcriptional lvl, the most efficient mechanism w minimal wastage
- More varied proteome despite limited genome size: by alternative splicing
How does histone acetylation regulate gene expression?
Histone acetylation, catalysed by histone acetyltransferase: add acetyl grps to (lysine residues) histones→ removes +ve charge on histones, ↓ electrostatic interactions between -vely charged DNA and histones→ chromatin decondense
RNA pol & GTFs can access and bind to promoter, assembles transcription initiation complex→ increase/initiate transcription
How does histone deacetylation regulate gene expression?
Histone deacetylation, catalysed by histone deacetylase: remove acetyl grps→ restoring +ve charges on histones, restores tighter interaction/↑ electrostatic interactions→ chromatin condenses
RNA pol & GTFs cannot access and bind to promoter (blocked) → prevent formation of transcription initiation complex→ prevent transcription
How does DNA methylation regulate gene expression?
DNA methylases add methyl grps to selected cytosine nt in specific CG seq
→ recruit DNA-binding proteins aka transcriptional repressors, histone deacetylase, repressive chromatin remodelling complexes to condense chromatin
→ block binding of GTFs, prevent assembly of …
RNA pol & GTFs cannot access and bind to promoter (blocked) → prevent formation of transcription initiation complex→ prevent transcription
How does CRC regulate gene expression?
CRC alters struc of nucleosomes temporarily: DNA less tightly coiled to histones → chromatin decondense
RNA pol & GTFs can access and bind to promoter, assembles transcription initiation complex→ increase/initiate transcription
How does repressible CRC regulate gene expression?
alters struc of nucleosomes temporarily: DNA more tightly coiled arnd histones→ chromatin condenses
RNA pol & GTFs cannot access and bind to promoter (blocked) → prevent formation of transcription initiation complex→ prevent transcription
How does adding 5’ cap regulate gene expression?
Adding 7-methylguanosine nt to 5’ end of pre-mRNA shortly after transcription begins
- Helps the cell recognise mRNA, so subsequent steps like splicing & polyadenylation can occur
- Acts as a signal to export mRNA out of nucleus
- Stabilises and protects growing pre-mRNA from degradation by ribonucleases
- Recognised by Eu initiation factors, which bind to it→ help recruit mRNA to small ribosomal subunit→ promotes translation initiation
How does polyadenylation regulate gene expression?
3’ end of pre-mRNA cleaved downstream of polyadenylation signal (AAUAAA) → poly-A polymerase adds a long seq of adenine nt to 3’ end of pre-mRNA (poly-A tail)
- Acts as a signal to export mRNA out of nucleus
- Stabilises and protects growing pre-mRNA from degradation by ribonucleases→ more proteins made
- Interacts w Eu initiation factors and 5’ cap for initiation of translation
Describe the formation of translation initiation complex
When small ribosomal subunit, eukaryotic initiation factors & initiator tRNA form a complex, which binds to 5’ cap & poly-A tail→ mRNA circularises→ complex moves in 5’ to 3’ direction to locate start codon, AUG → binding of large ribosomal subunit completes translation initiation complex
Explain how the length of 3’ polyA tail affects the half-life of mRNA and how it affects gene expression?
mRNA stability/half-life determined by length of poly-A tail. Longer = longer time mRNA can be used as template to make proteins
How is mRNA degraded?
poly-A tail removed by ribonucleases in 3’ to 5’ direction until a critical length is reached→ trigger removal of 5’ cap & degradation from 5’ end too
How is the formation of translation initiation complex regulated?
- translational repressors bind to 5’ cap & UTR, 3’ UTR→ interferes w interaction between poly-A tail, 5’ cap, initiation factors & small ribosomal subunit
- activation/inactivation by phosphorylation/ dephosphorylation of eIFs→ affect availability of activated eIFs
- Anti-sense RNA, complementary to part of mRNA to be degraded, is synthesised & binds to mRNA, forming a dsRNA→ block translation of mRNA + dsRNA targeted for degradation by nucleases
Describe the gene regulation at the post-translational level
- Covalent modification/cleavage (eg glycosylation) of polypeptides→ functional proteins
- Phosphorylation/dephosphorylation of translation IFs (eIFS) activates/deactivate protein→ up/down regulate activity
- Proteins targeted for degradation tagged with ubiquitin (by enzyme ubiquitin ligase), recognised & degraded by proteasomes into small peptides
What factors increase the chance of cancer?
- Environmental factors: exposure to carcinogens (eg tar) and ionizing radiation (eg UV radiation) → mutations
- Loss of immunity due to infection with certain viruses: HIV weakens immune system, reduces body’s ability to fight infections by other viruses that can cause cancer
- Genetic predispositions: gene mutations inherited
- Age: increase age, increase chance of cancer due to accumulation of mutations in cell over a lifetime
Define proto-oncogene
normal gene coding for proteins (growth factors/activators/growth signal transduction factors) that stimulate normal cell division/proliferation
Explain what is meant by a oncogene
Gain-in-function mutations in proto-oncogene (only 1 copy)→ oncogenes
- excessive production of gene product that stimulates cell cycle
- ↑ intrinsic activity of gene product: hyperactive or more resistant to degradation
==> uncontrolled cell division
Define tumour supressor gene
normal gene coding for protein products that inhibit cell divisions / activate cell cycle arrest, DNA repair, apoptosis→ preventing uncontrolled cell division
Explain the role of p53 gene in preventing uncontrolled cell division
p53 gene codes for specific transcription factor (p53 protein), an activator, that activate/upregulate expression of genes involved in:
- Cell cycle arrest: time to repair damaged DNA & prevent formation of mutant daughter cells
- DNA repair: prevent mutations that forms of oncogenes/inactivated tumour suppressor genes
- Initiating apoptosis when DNA is beyond repair: removes cells w damaged DNA & potential for cancer
How does loss-in-function mutation lead to uncontrolled cell division?
Loss-of-function mutation by point mutation/chromosomal translocation/retroviral integration (both copies)→ inactivate gene→ cell cycle continues wout repairing DNA/apoptosis/inhibition of cell division
Compare gain-in-function mutation and loss-in-function mutation
Definition
Gene that mutation affect
Type of mutation and no. of allele to be mutated to be cancerous
Effect on cell cycle in cancer cells
What makes a tumour malignant?
Invasive, can metastasise
How does a primary tumour form a secondary tumour?
primary tumour breaks through basal lamina, invades capillary→ cancer cell adhere to blood vessel walls, escape from blood vessel→ proliferate to form secondary tumour
Compare normal cells and cancer cells
Genes Cell division Nuclei Apoptosis Cells Contact inhibition Differentiation Cell adhesion / metastasis Angiogenesis
Why is cancer a multi-step process?
- Accumulation of mutations in genes which control regulatory checkpoints of cell cycle, in a single cell→ disrupts normal cell cycle→ cell has excessive cell growth and proliferation→ uncontrolled cell division
- (criteria 1) Gain-in-function mutation is a dominant mutation where mutation in just one copy of a proto-oncogene→ overexpression→ production of excessive amounts of growth factors; production of hyperactive/degradation resistant growth factors
- (criteria 2) Loss-in-function mutation is a recessive mutation, mutation in both copies of a tumour suppressor gene will disrupt their ability to inhibit cell cycle, enable DNA repair and promote apoptosis.
- (criteria 3) Activation of genes coding for telomerase→ telomeres lengthened, cell can divide infinitely
- Loss of contact inhibition→ cells grow into benign tumour
- Angiogenesis within the tumour→ blood vessels formed can transport oxygen and nutrients to it for its growth & remove metabolic waste products and CO2 → form malignant tumours that metastasise via bloodstream & form secondary tumours there.
