Biochemistry Flashcards
What is a pseudogene?
a section of a chromosome that is an imperfect copy of a functional gene
What are some of the functions of junk DNA?
at least 80 % of DNA serves some kind of purpose apparently
- places for proteinsto attach to that infuence gene activity
- strands of RNA with various roles, influencing gene action - non coding
- Places where chemical modification (backbone/methylation/phospharylation) silence stretches of chromosome
Which base is different in RNA?
Uracil instead of thymine
What sugars are in the backbone of DNA and RNA?
DNA - deoxyribos
RNA - ribox
What is the coding strand and the template strand?
Genetic info carried on the coding strand and not the template strand - template strand used for replication of DNA
Where does RNA polymerase attach?
Binds to one or more short sequences upstread of the start of each gene i.e. slightly closer to the 5 prime end (these are the promoter sequences of DNA)
What are the 3 types of RNA polymerase? -
Polymerase I - nucleolar region of nucleus, transcribes
large ribosomal RNA
Polymerase II - mRNA precursors (mostly producing the mRNA)
Polymerase III - small RNAs (tRNA), 5S ribosomal RNA
and other small DNA sequences
What is RNA polymerase composed of?
Several subunits and requires several accessory proteins (transcription factors).
All added to the complex in a defined order to initiate and carry out transcription.
What are basal promoters?
basal promoter contains TATA box and found in all protein-coding genes
What are enhancers?
DNA sequences which can control efficiency
and rate of transcription. Regulate expression of genes in
specific cell type and control timing of gene expression.
Effects can be powerful
Why are promotors and enhancers cis acting elements?
they are on the same molecule of DNA as the gene they regulate
What can changes in promoter strength, deleterious effects on a cell result in?
disease e.g tumours
Where can enhancers be placed?
can be 5’ or 3’ of transcription start site, in introns or even on non-coding strand
Can be thousands of nucleotides away from promoters with which they interact, brought into close proximity by looping of DNA (due to interactions between proteins bound to enhancer and those bound to promoter)
What are activators and repressors?
Protein facilitating looping are called activators and those that inhibit are repressors
How do enhancers affect Transcription factors?
Enhancers contain binding site sequences for transcription factors (TF) and enhance/upregulate transcription.
Active enhancers are bound by activating TF and brought into proximity of target promoters by looping
What are transcription factors?
Transcription factors bind to promoter and enhancer sequences
and recruit RNA polymerase
Why are TF trans acting factors?
they are encoded by a different gene to that being regulated
What does the polymerase enzyme do to the DNA?
it unwinds the double
helix over a short length and splits them apart – “bubble” of
about 10 bases
How can RNA become functional?
base-pair interactions between complementary sequences found elsewhere on same molecule allow an RNA molecule to fold into a three-dimensional structure that isdetermined by its sequence of nucleotides - similar to protein folding, this allows it to have structural and catalytic functions
How is polymerase 1 terminated?
hair pin loop which causes RNA pol to pause and release transcript.
How is polymerase 2 terminated?
Transcription of pol II genes can continue for hundreds or thousands of nucleotides beyond the end of a coding sequence.
Mature pol II mRNAs are polyadenylated at the 3’ end
= poly(A) tail (AAAAAAAAAAA).
- signifies end of RNA sequence
What does the CAP on the 5 prime end do?
stabilize the mRNA,
essential for transport of RNA out of nucleus
PROTECTS RNA FROM DEGRADATION
Serves as assembly point for proteins needed to recruit
small subunit of ribosome to begin translation
What is altenative splicing?
Different parts (introns and exons) of the same sequence can be removed to make different sequences i.e. one exon may be removed in one sequence but kept in in another so different proteins produced from the same gene by alternative splicing
- gives us diversity
alternative selection of splice sites within a pre-mRNA - leads to production of different mRNA isoforms of a gene
- alters composition and function of encoded protein
- plasticity allows for disease development –cancer
What is a spliceosome?
The enzyme that does the splicing
What are the 2 types of spliceosome?
Major – removes 99.5% of introns
Minor – removes remaining 0.5%
What are the 2 main functions of a spliceosome?
- Recognition of intron/exon boundaries
2. Catalysis of cut and paste reactions which remove non-coding introns and stitch flanking exons back together
How can spliceosomes cause disease?
Mutations altering splice site or spliceosome proteins
Mis-splicing = rapid degeneration of mRNA
Mis-regulation of splicing factor levels = cancer
What are the types of RNA?
mRNA
rRNA
- tRNA
- Non-coding RNA (ncRNA)
- Small nuclear RNA (snRNA)
- small nucleolar RNA (snoRNA)
- micro RNA (miRNA)
What does rRNA do?
Build ribosomes, the machinery for synthesizing proteins by translating mRNA
What are the 4 types of ribosomes?
18S - one of these along with other proteins
make the small subunit of the ribosome.
28S, 5.8S and 5S - one each of these, along with 45 other
proteins used to make the large subunit of the ribosome
(how dense)
How many different kinds of tRNA are there?
32 kinds
What can non coding RNA do?
ncRNA can modify protein levels by mechanisms independent of
transcription.
ncRNA play major roles in cellular physiology, development,
metabolism and are implicated in disease process.
What type of RNA are part of spliceosome?
Small nuclear RNA
What do snoRNA do?
Participate in making ribosomes by helping to cut large
precursor of 28S, 18S and 5.8S.
Modify many nucleotides in rRNA, tRNA and snRNA e.g. can
add methyl groups to ribose
Implicated in alternative splicing of pre-mRNA
Template for synthesis of telomeres
In vertebrates snoRNAs made from introns removed during
RNA processing.
What does micro RNA do?
Tiny RNA molecules regulate gene function post-transcriptionally - estimated more than one third of protein-coding genes are under control of microRNAs
very small - 18-25 nucleotides
Binds to mRNA and causes degradation - inhibits protein synthesis
Regulation of developmentally timed events.
Exhibit tissue-specific and/or developmental-
stage-specific expression.
Cancer development
microRNA’s apparently play a critical role in tooth development
What are the essential amino acids from diet?
Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Typtophan Valine
What are non-essential amino acids?
Alanine Aspargine Aspartate Glutamate Serine
Which amino acids are conditionally essential? (more needed in certain cirumstances)
Arginine Cysteine Glutamine Glycine Proline Tyrosine
How can non-essential amino acids be made?
9 non-essential amino acids are made from glucose + N source (amino acid or ammonia)
Non essential amino acids can also be made from essential amino acids:
- Methionine donates S for cysteine
- Phenylalanine forms tyrosine
Why do amino acids have to be degraded?
excess can neither be stored or excreted
Sources of excess amino acid?
diet exceeds need
some amino acids no longer needed during normal synthesis and degra
What can happen during fasting?
Proteins in the muscle can act as an energy source and be broken down into amino acids
What are glucogenic amino acids converted to?
their carbons are converted to glucose
What are ketogenic amino acids converted to?
acetyl CoA or acetoacetate (ketone bodies)
Where are amino acids broken down?
Liver
What is the nitrogen of amino acids used to form?
formation of ammonia then fed into urea cycle
What is transamination
Removal of nitrogen from amino acids
Amino group from one amino acid transferred to another
α ketoglutarate and glutamate usually one pair
Cofactor involved - pyridoxyl phosphate (derived from vit. B6 )
Reaction reversible- involved in synthesis & degradation
What can glutamate do?
Glutamate can collect nitrogen from other amino acids
What converts the nitrogen to ammonia for it to then enter the urea cycle?
glutamate dehydrogenase
What does ubiquitin?
Small proteins that targets protein for degradation
What is a proteasome?
protease complex where protein is unfolded & degraded (ATP)
What is the useable form of nitrogen?
ammonia Nh3
ammonium nh4+ toxic
What do we use to get rid of nitrogren?
Urea (urea cycle)
What is nitrogen balance?
nitrogen ingested (dietary proteins) = nitrogen excreted
Where do the urea cycle steps occur?
1st two in mitochondrion, other 3 in cytosol
What forms can nitrogen enter as?
amino acids - ammonia (transamination)
or as aspartic acid
Which amino acid is initiating and regenerating
Ornithine
What type of regulation is in the urea cycle?
feed forward - higher the rate of ammonium production, higher the rate of urea
What sort of activation of enzymes in the urea cycle?
Allosteric activation
What induces urea cycle enzymes
High protein diet or fasting (protein in muscles being broken down into amino acids which need to get rid of) induces urea cycle enzymes
Where is alanine formed?
From pyruvate in the muscles
What are alanine and glutamine used for?
These are broken down to glucose and ketone bodies in the liver & used for energy
What are ketone bodies?
Combo of Acetoacetate, beta hydroxybutyrate
Reconverted to acetyl CoA and enters TCA cycle for energy when glucose low
Acetoacetate spontaneously breaks down to acetone
‘Fruity’ smell of breath in ketotic states
Ketone bodies are acidic- ketoacidosis
What is phenylketonuria?
mutation in phenylalanine hydroxylase, mental retardation
What can urea cycle disorders lead to?
accumulation of ammonia, toxic to the nervous system
What are variable number of tandem repeats?
repeating nucleotide sequences - number of repeats and loci are inherited from parents.
Large variation of number of these regions between individuals.
- run according to size in the gel
How can you produce variable number of tandem repeats?
PCR - measuring transcription of gene
mRNA reverse transcribed to DNA then use PCR to detect lots of copies of the DNA
What do you need for PCR?
template dna
A, C, G, T
primers
magnesium and Taq polymerase
How does PCR work?
step 1 denaturation - heat to break hydrogen bonds between chains to open them up
step 2 annealing
primers sticking to specific base sequences of the template DNA at the beginning and end - 55 ish degrees
- enzyme added at 72 egrees
Step 3 - extension
base pairs join onto the template strand in the middle to complete copy of strand
Problems with PCR?
Poor precision Lower sensitiity Short dynamic range low resolution non-automated size based discrimination only results not expressed as numbers staining is not quantitive
What does real time PCR allow?
Gives you kinetics of reaction - quantifies it
What is the exponential phase?
The optimal point for analyzing data
How can DNA in PCR be identified?
Tags attached to the DNA ives of fluorescent dye picked up by machine to tell you new DNA piece been made
What does SYBR green bind to?
Any double stranded DNA
What are the advantages of real time PCR?
- Traditional measure at end point and real time collects data in exponential phase
- increase in reporter fluorescence directly proportional to number of amplicons generated
- increased dynamic range of detection
- no post PCR processing
- Detection down to 2 fold change
What is in situ hybridisation?
localisation technique for the detection of a gene product (RNA) in tissues - staining method using RNA as a probe
What is transcriptomics?
Genome- wide RNA transcript expression levels
What is proteomics?
large scale study of proteins partciularly their structures and functions, includes post translational modifications
What is metabolomics?
Comprehensive characterization of small molecule metabolites in biological systems.
What is a single nucleotide polymorphism?
DNA sequence variations when single nucleotide in genome sequence is altered
What are the 3 types of cell communication?
Remote signalling by secreted molecules
Contact signalling by membrane bound molecules
Contact signalling via gap junctions
What are the types of chemical signalling?
- Endocrine - hormone produced, enters bloodstream and is carried to target cell.
- Paracrine - local chemical mediator released, acts on cells in immediate environment (eg cytokine)
- Autocrine – on itself
- Synaptic - neurotransmitters released at synapses, diffuse to post synaptice target cell
What is the pathway to convert a signal to a response?
Signal - reception - transduction - response
What types of extracellular signals exist?
Growth factors Hormones Extra-cellular matrix Chemicals Proteins Sugars Synaptic
What type of cell responses exist?
Growth, cell division Differentiation Metabolism Apoptosis Gene transcription Secretion Contract / relax Membrane charge Migration
How can intracellular receptors cross plasma membrane?
Hydrophobic
How does phosphorylation affect cell responses?
Phosphate from ATP added
big charge changed - used as on/off signal
What type of membrane receptors exist?
G-protein linked
Tyrosine kinases
Enzyme linked
Ion channels
What do G-protein coupled receptors do?
Integral trans-membrane proteins
Receptor occupation promotes interaction with G-protein
Promotes exchange of bound GDP for GTP
activates G protein (α subunit) which leaves receptor
initiates signalling through secondary
messengers
What do receptor tyrosine kinases do?
Dimerise upon ligand binding
Have intrinsic enzymatic activity
Phosphate from ATP to tyrosine on itself (autophsophorylation)
Bind src homology-2 (SH-2) proteins
Initiate series of phosphorylation reactions
What do cytokine receptors do?
No intrinsic activity but associates with enzymes - JAKs
What do ion channels do?
Receptor is an ion channel
Ligand (eg neurotransmitter) binds to & opens channel (some are voltage gated)
Response: influx of Na+, change in membrane potential, action potential
What are secondary messengers?
Small molecules that bind and activate other molecules
cAMP
IP3
Ca2+
Diacylglycerol
How do FGFR’s stimulate fibroblasts and neuronal cells differently?
Fibroblasts - proliferation
Neuronal cells - differentiation
Which 3 amino acids can be phosphorylated?
Serine, threonine, tyrosine
How does phosphorylation of proteins allow it to react?
Charge change - change of shape so can react
What happens with Ras becomes insensitive to GTPase due to a genetic defect of the monomeric protein?
Uncontrolled growth - tumour/cancer
What do tyrosine kinases do?
Adds phosphate molecule to itself from ATP
What purpose do cascade systems serve?
Amplify the reaction and lots of opportunities to control the processes
What do cytokines receptors associate with?
Enzymes (JAKs)
What is cross talk?
2 different signals at cell
1 pathway can activate or inhibit the other pathway
E.g. integrin receptors and growth factor receptors
What is a scaffold protein?
Bring things closer together to make the reaction more likely or protect molecules from a reaction so it is less likely to occur
What does the cell cycle lead to the production of?
Two identical daughter cells - DNA duplicated exactly and divided equally
What happens in the S phase of the cell cycle?
Replication of DNA
What happens in the G1 and G2 of the cell cycle?
G1 - cell grows
g2 - cell prepares to divide
What did the Rao and Johnson experiment show?
Mitotic nuclei release mitosis stimulating factor for cells in all phases
What do cyclin dependent kinases do?
Phosphorylase proteins that control cell cycle, levels remain fairly constant, must bind to appropriate cyclin
What is the G0 phase
Cells that have permanently or temporarily left the cell cycle e.g. terminal differentiation -neurones
What does dysregulated cell growth lead to?
Cancer
What type of gene is p53?
Tumour suppressor gene
P53 blocks cell cycle if DNA damaged but if mutated allows cell cycle to continue with damaged DNA
What happens in reduced levels of p27?
Poor outcome in breast cancer
What does a greater number of cells in active mitosis indicate?
Healing/ growth/ repair
What does suprabasal mitosis suggest?
Pathology
What may apoptosis destroy?
Destroys cells that may be a threat - virus infected, immune, dna damage
What can signal a cell to begin apoptosis?
Withdraw of positive signals e.g. growth factors, hormones
Receipt of negative signals e.g. UV, death activators, hypoxia
What happens to apoptotic bodies?
Phagocytosed by other cells in the surrounding area
What does the intrinsic system depend on?
Integrity of mitochondrial membrane
If compromised, cytochrome C released - leads to cascade of caspase activation and eventually apoptosis
Onca gene - bel 2 binds to apaf 1 and prevents cytochrome C from forming so inhibits apoptosis
What are caspases?
Proteolytic enzymes - effectors of apoptosis
Present as inactive proenzymes (zymogen)
Activation cascade
What does HPV inactivate?
P53
What does EBV protein cause?
NasopharyngeAl cancer
What do apoptotic bodies appear as under H&E stains?
Very eosinophilic areas in the images
What is biological ageing?
Complex biological process in which changes at the molecular, cellular and organ levels results in a progressive inevitable and inescapable decrease in the body’s ability to respond appropriately to internal and/or external stressors
What are the characteristics of ageing?
Increased mortality
Increased susceptibility & vulnerability to disease
Changes in biochemical composition of tissues
Decrease in physiological capacity
Reduced ability to respond to environmental stimuli
What are some theories of ageing?
Galen (AD129- 199)
Changes in body humours beginning in early life
Slow increase in dryness & coldness of the body
Roger Bacon (1220-1292)
Wear & tear theory
Result of abuses & insults to the body
Good hygiene may slow process
Charles Darwin (1809-1892) Loss of irritability in nervous & muscular tissue
What are programmed theories of ageing?
Biological clocks
Purposeful programme driven by genes
What are non programmed theories of ageing?
Progressive random, accidental damage
Loss of molecular fidelity
Is the evolutionary theory of ageing programmed or non programmed?
Programmed - ageing genes
Is moleculer/cellular theory of ageing programmed or non-programmed?
Non programmed - free radical damage to molecules
increased frequency of senescence
Is the system theory of ageing programmed or non-programmed?
Non - programmed
Neuroendocrine alterations result in age related physiological changes
Immunologic function declines- decreased resistance to infection, cancer & increased recognition of self
What is hutchinson-guilford progeria?
Rare genetic disorder
Mutation in LMNA encoding nuclear envelope protein: lamin A
Affects RNA transcription & chromatin organisation
Lack of DNA strand rejoining after irradiation
Accelerated ageing (atherosclerosis)
Usually die by 13
What is werner syndrome?
Mutation in WRN, DNA helicase family
‘caretaker of the genome’: DNA repair and transcription
Baldness, hair and skin ageing, calicification of vessels, cancers, cataracts, arthritis, diabetes
Die by age 50
Central control of ageing?
How many times do normal cell approx divide?
50 times
What type of cells have no limit in dividing?
Cancel cells
What are telomeres?
DNA sequences
Protect the ends of chromosomes
Progressive shortening with age
What is telomerase?
Reverse transcriptase Stabilizes telomere length Telomerase activity in 90% tumours
How can lifestyle affect ageing?
Leading a sedentary lifestyle may make us genetically old before our time (BBC news Jan ‘08)
Twins who were physically active during their leisure time appeared biologically younger than their sedentary peers.
Telomeres shortened more quickly in inactive people.
Could signify faster cellular ageing.
Why does molecule ageing occur?
Intrinsic thermodynamic instability of biomolecules
3D structure cannot be maintained
Conformational change, aggregation, precipitation, amyloid formation
Ageing: catabolic chance driven?
How does molecule ageing occur?
Free radicals
Accumulation of oxidative damage in proteins & DNA
Damage to mitochondrial DNA:
e- leak from e- transport, form free radicals leading to more DNA damage
Flies expressing superoxide dismutase (free-radical scavenger) live longer
Antioxidants to counter ageing (Vit C, E, β-carotene, 2-deoxy glucose)
What is extrinsic skin ageing?
Wrinkles, pigmented lesions etc.
Sun exposure, air pollution, alcohol, poor nutrition
Smoking- increase in metalloproteinase enzymes which break down collagen
What is calorific restriction and why may it lead to living longer?
Reduced oxidant production by mitochondria- less ROS damage
Induction of SIRT1- key regulator of cell defence
Increased protein turnover- lack of accumulation of damaged protein
Inhabitants of Okinawa, Japan: 40% fewer calories, longest lifespan & highest % of centenarians
What are oncogenes
Genes that code for growth factors etc that promotote autonomous cell growth and proliferation
What are tumour suppressors genes?
Code for factors which control the cell cycle, regulate apoptosis, transcription and cell interactions
Reduces cell proliferation and maintains tissue integrity
Defects - cell grows too fast and dies
What are mismatch repair genes?
Genes that code for enzymes that repair damaged DNA
What are factors in carcinogenesis?
Genetics factors
Environmental factors
Chemicals
Viruses
What are the two stages of chemical carcinogenesis?
Initiation - permanent DNA damage
Promotion - may be reversible, promotes proliferation
What is the latent period?
Time from initiation to clinical tumour
What is a pro-carcinogen?
Chemicals often metabolised to ultimate carcinogen (not carcinogen initially) - body activates it
What is a co-carcinogen?
Chemical not a carcinogen but when combined with a carcinogen it increases its effect
What is direct carcinogenesis?
Tumour arises at the site of carcinogen application e.g. smoking and lung cancer
(Indirect - tumour arises at different site from carcinogen application) e.g. aromatic amines
What is mesothelioma?
Cancer of the plural lining of the lungs
What type of tissue is the most sensitive to radiation?
Embryonic tissues
What is the most common cancer?
Basal cell carcinoma
How do cancers develop?
May be de novo - just appear e.g. salivary gland tumours
Via a benign tumour - adenocarcinoma of colon
Via a premalignant lesion - HNSCC
What is dysplasia?
Cellular abnormalities and disorganisation of the tissue before invasion
What are stem cells?
Undifferentiated cells which have the potential for self renewal and can give rise to one and sometimes many different cell types
What is used to deliver a gene to patient’s target cells?
Vector from a virus carries the gene inside to the target cell
What is CRISPR-Cas9? (Fault gene replacement system)
CRISPR finds the relevant gene
Cas-9 - cuts gene
Replace with donor gene
