genetics Flashcards
State the 3 stages at which gene expression can be regulated.
1) transcription
2) splicing
3) translation
Define transcription.
creates mRNA to allow the coding in DNA to be passed out of the nucleus
In transcription, what are mRNA, tRNA, rRNA are all created by?
RNA polymerase
State the role of each:
a) RNA Polymerase I
b) RNA Polymerase II
c) RNA Polymerase III
a) production of large ribosomal RNA (rRNA)
b) production of mRNA
c) production of tRNA & small ribosomal RNA molecules
State the 7 stages of transcription.
- start of gene of DNA is marked at the promoter region
- RNA polymerase molecules randomly collide with DNA in the nucleus. They bind with specific DNA sequences called promoters. (e.g. TATA sequence/box)
- RNA polymerase breaks the H bonds betw/ the complementary BPs in the DNA helix exposing both sides of the DNA strand
- complementary base paring takes place on DNA template strand – creating a single RNA strand
- the nucleotides are joined together by RNA polymerase
- mRNA unwinds from DNA strand
- mRNA exits the nucleus via the nuclear pore
What forms during transcription and only in which case does it occur?
- a transcription bubble It
- occurs when only a limited amount of DNA is unwound from the DNA helix
As a part of RNA processing, during transcription, how are the ends of the mRNA is capped at the 5’ end?
- addition of methylated Guanine nucleotide, by the removal of a phosphate by phosphatase
- addition of GMP (Guanosine monophosphate) via guanylyltransferase
- addition of methyl group via a methyl transferase
- allows the mRNA to exit the nuclear membrane
As a part of RNA processing, during transcription, how are the ends of the mRNA is capped at the 3’ end?
a tail of up to 200 nucleotides is added by poly-A polymerase.
What is an intergenic region?
non-coding piece of DNA
Explain how a gene and an intergenic regions (introns and exons) are edited before splicing.
- exon/gene region; codes for proteins; exits the nucleus
- introns are removed
- exons can also be removed if they are not the particular piece of DNA which is needed for that expression - protein
- pre-mRNA to mRNA
What is splicing mediated by?
the spliceosome
State the 7 stages of splicing.
- it consists of several protein-RNA complexes
- snRNPs and spliceosome bind on the RNA
- RNA is looped, more complexes bind
- the complex undergoes a conformational change
- intron is cleaved at 5’ end
- intron is cleaved at the 3’ end
- 2 exons are joined together
What is alternative splicing?
Use of other proteins to aid the splicing process. Allows a single gene to produce multiple varieties of protein.
What are the 5 possible ways alternative splicing can be done?
1) exon skipping – only including some exons and leaving some out.
2) mutually exclusive exons- taking either of the 2 consecutive exons in between
3) alternative 3’ acceptor sites – the splice junction at the 3’ end is used, changing the 5’ boundary of the downstream exon.
4) alternative 5’ donor sites – the splice junction at the 5’ is used, changing the 3’ boundary of the upstream exon.
5) intron retention – the intron is retained; stop codons
NB: (3) and (4) are opposites
State the 4 main characteristics used to code gene structure.
- promoter
- transcriptional ‘start’ and ‘stop’ signal
- exons and introns
- upstream regulatory regions
What does transcriptional control of genes usually involve?
- switching a gene on (promoter as genes are not always expressed)
- e.g. some genes may be expressed in response to stimuli or during a point in a cycle
What drives gene expression/
RNA polymerase II
What must bind in the promoter region in order for a gene to be expressed?
a number of DNA-binding proteins (transcription factors)
How is gene expression fine-tuned?
- via the binding of other transcription factors
- to distal regions termed upstream enhancer sequences
What do activators do when they bind, to enhancer sequences?
increase expression significantly than without them
How can a gene be switched off?
- transcriptional repressors
- not expressing a gene
- bind to specific sites on DNA and prevent transcription of nearby genes
How do the 2 types of repressors act?
1) interacts w/ a transcriptional activator (enzyme) - binds to the site next to the activator so that its function is blocked
2) overlapping binding sites - the repressor binds at the site where the activator would bind to, so the activator cannot bind
Give an example of a transcriptional repressor and explain how it works.
- Wilm’s tumour protein (WTP)
- in a developing kidney, WTP binds to the promoter region of the EGR-1 gene (a transcriptional activator) – switching off its expression
- if the gene encoding WTP is mutated, it leads to uncontrolled expression of EGR-1
- this can lead to development of kidney tumours in early life
- its considered a tumour suppressor gene
a) Write a piece of DNA which is 15 base pair long.
b) Make an RNA molecule out of the above DNA. – using 3’ to 5’ DNA strand
a) 5’-ACTGAGTACCTTTCG-3’ 3’-TGACTCATGGAAAGC-5’ b) 5’-ACUGAGUACCUUUCG-3'
State teh 3 main stages of translation (protein synthesis).
- tRNA carrying a methionine (start codon) associates w/ a small ribosomal unit which is in association with eukaryotic initiation factor 2 (eIF2)
- the ribosomal unit recognises the 5’ capped end of mRNA with 2 additional initiation factors (eIF4G and eIF4E)
- the ribosome scans along the mRNA to find the start codon; this allows the large ribosomal subunit to bind
What is translation elongation driven by?
elongation factors EF1 and EF2
State the 4 stages of translation elongation.
- tRNA (aminoacyl) binds to A site. tRNA molecule at E site is released.
- The carboxyl end of the amino acid in the polypeptide chain is uncoupled from the P site on tRNA. it then is attached to the new amino acid (on the tRNA molecule at the A site) by a peptide bone - peptide transferase enzyme.
- the large ribosomal unit steps one codon along the mRNA to carry on reading the code.
- The small ribosomal unit steps 1 codon along so that there is a new tRNA at each site. generating a new A site.
What happens during translation termination?
- protein synthesis is stopped when the ribosome encounters a stop codon (UAA, UAG, AGA)
- cytoplasmic release factors bind to the stop codon. This frees the carboxyl end of the growing polypeptide chain.
How is translational control implemented/
- many ribosomes attach to each mRNA
- in time mRNA is degraded (due to its half-life)
- the half-life is a way in which the cell can regulate gene expression levels
What are isoforms?
- proteins which have similar functions and similar amino acid sequences, but not the same
- this can arise from alternative splicing of a single gene
What can errors in gene expression result in and what can understanding these errors allow?
- uncommon disorders (DMD, Spinal Muscular Atrophy, Cystic Fibrosis, Pulmonary Arterial Hypertension)
- influence predisposition of many common diseases (Breast cancer, lung cancer, prostate cancer & blood cancer)
- understanding of these errors can help to develop therapies
For each gene mutated state the genetic disease caused:
a) DMD
b) SMN1
c) CFTR
d) BMPR2
e) BRCA1 +BRCA2
a) duchene muscular dystrophy
b) spinal muscular atrophy
c) cystic fibrosis
d) pulmonary arterial hypertension
e) breast cancer
What can over-expression of transcription factor (TF) MYC cause?
- numerous cancers
- if even 1 copy of the TF is mutated, it can still lead to disease
What is haploinsufficency ?
- where 1 copy of the gene is not enough (e.g. p53 gene)
- we have 2 alleles for each gene (mother & father)
- one of them can mutate, and one of them can stay the same. - one copy of the gene is not enough for protection
What is a dominant mutation?
- where a change in the gene sequence generates change in the protein that exerts dominance over the wild-type (phenotype)
- there is a change in AA and therefore a change in the protein produced
- i.e. TPCP2L3 gene & genetic deafness
Describe an example of mRNA processing errors that occurs during alternative splicing.
- cystic fibrosis
- mutations found in exon 7 of SMN1 gene
- exon 7 is skipped
- non-functional protein is produced.
- children only live for a few years
Describe example of mRNA translation errors that occurs during alternative splicing and state the type of TF and gene that are affected leading to each disease:
a) VWM – neurodegenerative
b) gastric cancer
c) rare types of anaemia & neurodegenerative diseases
- these mutations affect translational efficiency (mRNA into protein)
a) - translational initiation factors; eIF2
b) - release factors; eRF3
c) - N/A; affect tRNA transfer proteins/ ribosome itself
What are the techniques which detect DNA, RNA & proteins mutations used to do?
see the exact point where the mutation occurs
What is gel electrophoresis of DNA/RNA?
- lithium bromide binds to the DNA
- DNA is negatively-charged so will move from the anode (-) to the cathode (+)
- through the current passing through the (argarose) gel
What is western/southern blot used to detect and what are its 6 stages?
- detect proteins
1. sample preparation
2. gel electrophoresis
3. blotting (or transfer)- to a membrane
4. blocking
5. antibody Probing – attaches specifically
6. detection- labelled with dye or fluorescent markers
How can RNA be artificially created by PCR? State the method.
- RNA polymerase; DNA TO RNA
- for this to occur, a promoter region is needed on DNA to allow enzyme to attach and initiate the process
- free nucleotides are also needed
Describe how the splicing of pre-mRNA in a tube occurs.
- alternating splicing
- machines detect it
1. combine RNA with cytoplasmic extract (contains tRNA and ribosomes)
2. RNA to protein
How can PCR be used to detect gene expression?
- enables amplification of a specific region of DNA from a single molecule of starting material
- DNA polymerase is needed
- hydrogen bonds broken by heat
- from 5’ to 3’ the nucleotides are added to the parent strand by the aid of DNA polymerase
How can gene expression profiling be done by PCR?
- quantitative real-time PCR (qPCR)
- detects how many copies of a gene you have
- insight into regulation of gene expression at a given time
State 3 uses of PCR.
- diagnostics
- gene cloning
- legal disputes
What are nucleotides the building blocks of?
- DNA: deoxyribonucleic acid
- RNA: ribonucleic acid
- covalent bonds
State the 3 components of a nucleotide and state how they differ in DNA and RNA.
• Pentose sugar: - deoxyribose (DNA) - ribose (RNA) • Phosphate group: - acts as a bridge between adjacent ribose/deoxyribose groups • Nitrogenous base: - pyrimidine (C, T (DNA), U (RNA) - purine (G, A)
Which type of bond links a ribose molecule to a nitrogenous base?
a glycosidic bond
What is the rule of base-pairing in DNA and RNA.
- a pyrimidine always pairs with a purine
- i.e. C-G and T-A
- A-U bonding occurs during gene transcription (RNA)
Which bonds are formed between nucleotides and is aG-C bond or an A-T bond stronger and why?
- hydrogen bonds a
- G-C bond is stronger than A-T due to more H bonds (3-2)
State the structural features of the DNA double helix.
- hydrophobic bases are on the inside of the strand, away from the water stabilised by H-bonds
- hydrophilic-sugar-phosphate backbone is stabilised by electrostatic interactions & H-bonding with water
- bases which are stacked have weak transient (temporary) electrostatic interactions VDWF (pi-pi interactions)
What are the structural features of RNA and what can single-stranded RNA molecules form?
- stem: complementary base pairing
- loop: no base pairing
- can form secondary structures through base-pairing
How do DNA and RNA polymers differ in size?
- difference is the bases (monomer units) – U and T
- polymer is described by the sequence (of bases) and number of bases
- e.g. size is the number of monomeric units (X bases long)
How do DNA and RNA polymers differ in direction?
- nucleotides always add to the 3’ end
- the α-phosphate of new nucleotide attaches to the -OH group of polymer
- this forms a 3’-5’ phosphodiester bond
- chain grows in 5’ to 3’ direction (left to right)
- nucleic chain starts with 5’ phosphate and ends with 3’ OH
• e.g. 5’ACTGCT’
(see document for diagram)
Describe DNA organisation in a eukaryotic cell.
- to compact the DNA, it is held in the nucleus with the aid of proteins
- the DNA content in a human genome is much larger than the cell nucleus so its compacted to fit in the nucleus, whilst still being functional
- histone is a protein which is in chromatin
- linker DNA is double-stranded DNA in betw/ 2 nucleosomes
- H1 protein binds to the linker DNA, holding everything together
What is a:
a) chromatosome?
b) nucleosome?
c) chromatin?
a) nucleosome + histone H1
b) basic unit of compacted DNA
c) histone-bound DNA
Sort in order of complexity:
- chromsome territories
- nucleosome
- DNA duplex
- chromosomes
- chromatosome
DNA duplex → nucleosome → chromatosome → chromosomes → chromsome territories
What is core DNA?
(146 BP) wraps around the histone core
What is linker DNA?
(60-80 BP) leads to adjacent nucleosome
What does a histone core/octamer contain?
2 copies of 4 histone proteins:
- H2A
- H2B
- H3
- H4
Dseribe the histones and DNA backbone in an octamer.
- high Arg and Lys content (+ve charge)
- which allows them to bind to the DNA backbone (-ve charge) easily
What does DNA replication involve, when doe sit occur and what is it vital for?
- involves replication enzymes and RNA
- occurs in S phase of interphase during cell cycle
For which 3 things is DNA replication vital for?
1) cell growth
2) repair
3) reproduction
What does the semi-conservative model of DNA replication describe?
when each daughter strand contains ½ of parent strand
State and describe the 3 stages of the mechanism of DNA replication.
- formation of replication fork
- base pairs are broken by DNA helicase
- unzips double strand
- the strands form a Y shaped replication fork – template for replication
- proteins bind to this replication fork to stabilise this area so the single stranded DNA does not join back together
- 5’ to 3’ replication - RNA primer binding (leading strand)
- a short piece of RNA (primer 3-4 BP) binds to the 3’ end of the parent strand
- this marks the starting point for replication
- primers are generated by DNA primase - Elongation (leading strand)
- DNA polymerase α binds to the parent strand at the site of the primer
- this enzyme adds new complementary BP to the parent strand
- after 20 BP, elongation is taken over by DNA polymerase ε
- DNA polymerase ε and 𝛿 = proofread 3’ to 5’ exonuclease activity. This prevents incorporation of incorrect nucleotides.
- produces 1 continuous DNA strand in the 5’ to 3’ direction - termination (both strands)
- RNA primers are degraded by RNAse H
- RNA primers are filled by DNA polymerase 𝛿
- DNA ligase joins any breaks in both strands; continuous strand
(see document for diagrams)
What is a bi-directional replication fork?
- one strand is orientated in the 3’ to 5’ direction on the parent strand (leading strand)
- the other is orientation 5’ to 3’ on the parent strand (lagging strand)
(see document for diagram)
Why does DNA replication occurs at multiple sites?
- replicate cannot originate from 1 site per chromosome – it would take too long
- 1 genome takes 8 hours to replicate
- there are multiple origins of replication with replication forks proceeding in different directions – replication bubbles
What is a replication bubbles?
- there are multiple origins of replication
- with replication forks proceeding in different directions
(see document)
How are replication bubbles joined?
by DNA ligase
Describe the inheritance of histones after replication.
- histones are removed after DNA replication (in front of replication bubble) – they are not needed as there only purpose is to compact the DNA
- H3/H4 tetramers (histone octamer) remain intact
- new H3/H4 tetramer cores bind
- new H2A /H2B bind
What is epigenetics?
- heritable changes in the phenotype/cell behaviour or gene expression in cells
- caused by changes other than changes in DNA base sequence that control the activity of genes
Give examples of epigenetics.
- histone modifications (acetylation of Lys, methylation of Lys and Arg)
- DNA modifications (methylation of cytosine)
What do epigenetic modifications alter and why?
- alter chromatin structure to control accessibility of transcription factors
- and co-activators necessary for gene transcription
What can epigenetic marks can be altered by and what does this provide a mechanism for?
- environmental stimuli (smoking, nutritional status)
- provides a mechanism for environmental factors to be imprinted genetically
Explain the Dutch famine 1994 example of epigenetics.
- foetuses exposed to famine during 1994-45
- 50 years later less DNA methylation on the IGF2 gene compared with unexposed same sex siblings
- children developed obesity and schizophrenia later in life
- children born to these women 20-30 years later suffered from same problems – even though they were conceived and born in a normal dietary state
- the exposure took place after conception, therefore it suggests this period is important for establishing epigenetic marks that persist in generations
What can early life environmental conditions cause?
epigenetic changes that persist through life
State the mechanism for PCR.
- to amplify a single DNA strand into billions of identical copies
1. heat reaction mixture to 90 degrees. This denatures the DNA strands, breaking the H bonds between the complementary nucleotides.
2. temperature is reduced to 60 degrees. DNA primers anneal to their complementary base pairs.
3. temperature raised to 72 degrees. Taq polymerase attaches at the primer and starts to add the free nucleotides to the DNA strand.
4. 2 copies of DNA are now formed.
5. process is repeated many times, more identical strands are produced
Describe the stages of DNA 1st generational (Sanger Dideoxy) sequencing
- 4 test tubes are labelled A, T, C, G. into each tube goes:
- the sample of DNA which is to be sequenced
- DNA primer
- 4 DNA nucleotides (normal)
- DNA polymerase
- for each test tube, the dideoxy nucleotide is added. This is a nucleotide which has no OH on the 3’ end, therefore DNA synthesis is prevented. (tube A which contain dideoxy A etc.) - after many cycles, DNA polymerase synthesises many copies of the DNA sample. The lengths are varied as once the dideoxy-nucleotide has been added, DNA synthesis is terminated e.g. in tube A, all DNA sequences will end in A etc.
- contents of tubes are denatured and are run in an electrophoresis gel. This tells us the length of the DNA strands. Shorter DNA strands travel faster through the gel than the longer ones.
- dye fluorescence is added – detected by computer programme to reveal the sequence
Describe improvements in genome sequencing and what they will allow.
- ongoing improvements in DNA sequencing technology & data mean that individual genome sequencing will eventually be affordable
- cost will be reduced the same as a sophisticated diagnostic test
What is precision/personalised medicine?
influence of genetic variation on drug response in patients by correlating gene expression or presence of single-nucleotide polymorphisms (SNPs) with a drug’s efficacy or toxicity
Describe the reasoning behind precision/personalised medicine?
- optimises drug therapy in accordance to the individual patient
- to ensure maximum efficacy with minimal side effects
Give an example of precision/personalised medicine in drugs.
e.g. drugs/drug combinations and doses are optimised for each individual’s unique genetic makeup
Give an example of precision medicine in clinical practice.
- cohort created: 1 million American volunteers that share genetic data, biological samples, diet and lifestyle info all linked
- sequence 100,00 genomes of patients with cancers, rare disorders & infectious diseases
- link data to extensible account of diagnosis, treatment and outcomes
- produce new capability in genomic medicine for transforming the NHS
What is a polypeptide?
amino acid monomers linked together by peptide bonds
What is a protein?
> 40 AAs that can fold into a defined shape
State the 4 levels of protein structure.
- primary structure - AA sequence
- secondary structure - interactions betw/ adjacent AAs i.e. α helices, sheets, loops/random coils
- tertiary structure - 3D folding of single polypeptide chain
- quaternary structure - assembly of multiple proteins into complex
In what order is an AA sequence taken and what is a DNA sequence determined by?
- AA sequence from N-terminus to C terminus (left to right)
- determined by DNA sequence of gene for protein
- dictates final protein structure (sequential arrangement of R groups influences subsequent 2, 3 & 4 structures)
Give an example of a disease caused by a change in the primary structure of a protein.
- Example: Sickle cell disease
• caused by a single mutation in HbA haemoglobin gene
• single mutation in B-globin gene (T to A) changes 10 sequence (Glu to Val)
By which bonds is the overall 3-D shape of a protein held by?
- hydrogen bonds betw/ R-groups
- ionic bonds - electrostatic attraction betw/ CO2- and NH3+ of R Groups
- disulphide bridges - betw/ cysteine –SH groups; strongest as they are covalent crosslinks
- hydrophobic interactions - hydrophobic R-groups cluster
How long does it take for 3D confirmation of protein structure?
- attained within seconds
- small regions of relatively stable secondary structure are formed first
Which 2 proteins does tertiary folding results in?
1) fibrous proteins
2) globular proteins
What are proproteins?
inactive peptides/proteins that need post-translational modifications to activate them
Describe the stages of production of insulin as an example of tertiary folding.
- ribosomes feed growing AA chain (preproinsulin) directly into the ER
- signal peptide is present to direct chain to its right location; once it has reached it, it is cleaved off by signal peptidase as this is no longer needed. – producing proinsulin
- oxidation of -SH groups to -S-S (Disulphide bridges) are formed allowing chain to fold – (covalent bonding)
- C-chain is then cleaved & removed to create active protein
What can post-translational modifications and what they can involve.
- processing (proteolytic cleavage to an active form) AND/OR
- covalent modification (occurs after translation & is a chemical modification)
What happens at the start of translation?
- during translation, 20 genetically-encoded AAs synthesised
- PT covalent modifications extend structure & function
- changes in chemical structure change in spatial structure & biological activity
Are all PT modifications reversible and how is this used?
- some PT modifications reversible (i.e. acetylation, phosphorylation, methylation) – allowing rapid dynamic regulation of protein activity
- controlling PTM allows control of their activity
- principle widely-used in nature to regulate numerous biological processes i.e. metabolism
- catalysed by enzymes, involved in regulation of target protein’s activity
PTMs are key mechanisms in increasing what and how?
- proteomic diversity
- our genomes comprised of 20,000-25,000 genes
- our bodies can produce many more proteins (proteome) than this by changes at transcriptional & mRNA levels
- increases size of transcriptomes (something which is transcribed into genes)
- this increases number of functional proteins in our bodies
State and classify all of the post-translational protein modifications (structural changes).
• proteolytic cleavage (irreversible): - removed from N-terminus - removed from internal part • proline isomerisation • addition of small functional groups: - phosphorylation (reversible) - methylation (reversible) - acetylation (reversible) - hydroxylation - ubiquitination • changes in chemical nature of AA • addition of large functional groups & macromolecules: - glycosylation - addition of other peptides/proteins - addition of fatty acids & lipid residues: ○ C-terminal glycosyl phosphatidyllinositol (GPI) anchor (irreversible) ○ N-terminal myristoylation (irreversible) ○ S-myristomylation (irreversible) ○ S-prenylation (irreversible)
What is PTM proteolytic cleavage?
occurs at a peptide bond; either at:
- N-terminus
- internal part of protein
What is PTM proline isomerisation?
the change in the AA proline residue spatial confirmation; produces cis and trans versions
What is PTM addition of small functional groups?
- phosphate is donated by ATP to an acceptor protein
- catalysed by protein kinase
- serine is most commonly phosphorylated AA, followed by threonine
- tyrosine phosphorylation (e.g. 2) leads to binding of specific proteins which is part of a signalling network
(see document for diagrams)
What is de-phosphorylation and how is it catalysed?
- removes phosphate group from protein and gives it back to ADP
- catalysed by protein phosphate
Explain the example of protein phosphorylation with pyruvate dehydrogenase
- pyruvate dehydrogenase
- protein kinase is activated by high [NADH] : [NAD+] and [acetlyCoA] : [CoA]
- protein kinase is inhibited by pyruvate, so it can enter krebs cycle
How is an example of protein phosphorylation shown in the EGF (growth factor) pathway?
- EGF (growth factor) pathway
a. EGF binds to receptor
b. receptor changes shape – dimerization as the 2 receptors join together
c. undergo autophosphorylation at C terminus
d. phosphorylation recruits proteins to the receptor
How is an example of protein phosphorylation shown in the cell cycle?
- cell cycle is controlled by cyclins and their cyclin dependent kinases (CDKs)
- G1 and S phase (checkpoints)