DNA and diseases -> LECTURES 4-6 Flashcards
how much of DNA is non-coding
Approx 98.5-97%
what are the differences between DNA and RNA
- The sugar in RNA is ribose (Ribonucleic Acid), not deoxyribose
- Uracil is present in RNA (T in DNA)
- RNA molecules are shorter than DNA
- RNA is single stranded
- DNA has1 function: storing genetic information
- 3 main kinds of RNA each with different functions
What are the 3 main types of RNA
- Ribosomal RNA’s (rRNA)
- Messenger RNA’s (mRNA’s)
- Transfer RNA’s (tRNA’s)
what is rRNA
- Ribosomal RNAs (rRNA)
- Exist outside the nucleus in the cytoplasm in ribosomes: small, granular structures where protein synthesis takes place
- comprises >95% of total cellular RNA. This class of
RNAs are assembled together with numbers - ribosomal proteins, to form the ribosomes.
- Comprises of approx 2% of total cellular RNA.
what are ribosomes made up of
- Ribosomes: complex consisting of approx 60% rRNA and 40% protein
What is mRNA
- Messenger RNAs (mRNAs)
- “Record” information from DNA in the nucleus and carry it to the ribosomes
What is tRNA
- Transfer RNAs (tRNA)
- Deliver AA’s one by one to protein chains growing at ribosomes
What is Chromatin
uncoiled form of DNA and over 50% protein
What is a chromosome
coiled DNA/protein that form during the early stages of cell division
what is epigenetic regulation of genes
a control system that dictates which of many genes a cell
uses and which it ignores.
- switch genes on and off
- allows rapid response to environment without having to change DNA
what are the 3 main mechanisms of epigenetics and explain
- Addition of methyl groups (Methylation) to some regulatory regions of the DNA reduces gene transcription
- Small and reversible chemical modification to chromatin e.g. addition of acetyl groups (acetylation) to histones enhances transcription
- microRNAs: can degrade mRNA or regress translation
- Lifestyle, diet and exercise have an impact of genes
What is DNA replication
- The process of making new DNA
- New strand formed by pairing complementary bases with old strand
- 2 molecules made: one new and one old DNA strand
- Semiconservative - old/new and new/old
what direction is new DNA synthesised?
- only 5’ to 3’ formed (polymerase moves in 3’-5’ direction on existing strand)
- Leading strand synthesis in 5’ to 3’ direction (continuous)
- Discontinuous synthesis - 5’ to 3’ DNA segments
what are the enzymes involved in DNA replication
- Helicase (unwinds the double helix)
- Single strand binding proteins (stabilise separate strands)
- RNA Primase (adds a short RNA primer to DNA strand)
- DNA Polymerase (binds nucleotides to form new strands)
- Exonuclease (removes RNA primer and inserts correct base)
- DNA ligase (joins Okazaki fragments and seals other nicks in sugar-phosphate backbone)
Explain the process of DNA replication
- the primase uses the DNA strands as templates to synthesise a short stretch of RNA (primer for polymerase)
- RNA primer supplies a free 3’-OH which the polymerases can attach the 5’-phosphate of an incoming nucleotide to
- DNA polymerase catalyses formation of H bonds between complementary bases and catalyses formation of phosphodiester bonds between 5’-phosphae and free 3’-OH
why is it RNA primase for DNA synthesis/replication
primase is an RNA polymerase, not a DNA polymerase. There are no DNA polymerases that can initiate synthesis in the absence of a free 3’-OH group except RNA primase
how often is there an incorrect base in DNA replication
- 1 incorrect base per 10^4 to 10^5 bases
- highly accurate
what is meant my polymerase proofreading
- DNA polymerase can remove and replace incorrect bases
- Error rate 1 base per 10^8
what is the overall error rate in DNA synthesis repair
1 base per 10^10 bases
what is telomerase and telomere extension
- end of DNA -> shortens when cells divide
- telomerase binds to 3’ end of telomere sequence along with RNA template (use complementary RNA as template)
- catalyses addition of bases, restoring telomere length
What are the causes of mutations
- Errors during DNA replication
- Jumping genes/transposons - can cause deletion, insertion, frameshift and inversion - mutations
External factors:
- Chemical
- UV light
- Ionising radiation
what are the possible ways a mutation is expressed silently
- Mutation occurs in a gene that is not expressed by that cell
- Mutation alters only 1 copy of gene, and the other copy is still expressed as dominant
- Mutation occurs in non coding DNA
- Mutation does not alter amino acid sequence of the protein
Types of mutations
- Point mutation - change of base pair - replication mistake (missense, nonsense, repair mistake)
- Deletion mutation - removal of one or more bases - intecalating chemicals, DNA polymerase slips mobile genetic elements
- Insertion mutation - insertion of one or more bases - intercalating chemicals, mobile genetic elements
- Frameshift mutation - deletion or insertion of a number of bases
that cannot be divided by 3 - intercalating chemicals, mobile genetics - Inversion mutation - inversion of a sequence of bases (may cause frame shift) - mobile genetic elements
effects of point mutations in coding regions
- Silent mutations: codes for the same amino acid
- Nonsense mutations: codes for a stop, which can truncate the protein
- Missense mutations: codes for a different amino acid
What is Canine Progressive retinal Atrophy (PRA)
- An example of a nonsense mutation
- Degenerative disease in photoreceptor cells - with this disease, cells deteriorate over time leading to blindness in dog
- Progressive retinal atrophy (PRA)
- Rod-cone dysplasia type 1
- Point mutation in the cGMP phosphodiesterase b subunit gene
- Results in a stop codon within the coding region of the gene
- elevated levels of cGMP
- retinal cells degenerate, leading to blindness
what is Feline Polycystic kidney disease (PKD)
- An example of a nonsense mutation
- Most prominent inherited feline disease
Clinical signs:
- Increased drinking and urinating; diminished appetite, weight loss, nausea, vomiting, lethargy
- C>A point mutation in an integral membrane protein gene, polycystin-1 (PKD1) - results in stop codon
- Reduced PKD1 signalling in the primary cilia of tubular epithelial cells leads to cystic disease
- No treatment
- Selective breeding
What is anaemia in Basenjis
- An example of a frameshift mutation
- Deletion of single base in 433rd codon of pyruvate kinase (PK) gene
- Causes a shift in reading frame
- Misformed and shortened protein is unstable in RBC’s
- A new stop codon is ultimately encountered
- Lack of PK causes RBCs to slowly be destroyed - anemia No treatment
What is Von Williebrand disease (vWD)
- An example of a frameshift mutation - caused by point mutation
- Most common inherited coagulopathy in dogs
- Deficiency of von Williebrand factor (vWF)
- Blood glycoprotein, binds to other proteins
- G>A nucleotide substitution
- Activates a cryptic splice site
- Result: frameshift - formation of a truncated vWF (5-10% normal function) Autosomal recessive
No treatment - transfusion for severe bleeding
Where do chromosomal mutations occur?
- meiosis
types of chromosomal mutations
- Deletions: piece of a chromosome missing
- Duplications: piece of a chromosome appears twice
- Inversions: piece of a chromosome turns 180 degrees
- Translocations: a piece of a chromosome attaches to another chromosome
- Leukemia
- BCR/abl fusion protein is an overactive kinase
why can chromosomal translocation cause cancers
- Interchange of parts between chromosomes
- Places the coding region of a gene near promoter/enhancer region of another gene, leading to over-expression of first gene
- Major cause of some cancers
what is Canine Burkitt Lymphoma (BL)
- Approx 5% of canine lymphomas
- Region of canine chromosome 13 containing MYC oncogene translocation to region of chromosome 8
- Contains the immunoglobulin heavy-chain (IgH) enhancer
- Leads to MYC over-expression in B-cells - cancer
What is Diffuse large B-cell lymphoma (DLBCL)
- Most common form of canine lymphoma (approx 50%)
- BCL-6 transcription factor on chromosome 3 moves to IHG locus
- Resulting in over expression of BCL-6
- Constitutive BCL-6 expression in B-cells: cancer
What is transcription and process
- ranscription is the mechanism by which template strand of DNA is utilised by specific RNA polymerase to generate on elf the tjree different classifications of RNA: mRNA , tRNA and rRNA
- RNA Polymerase II “opens” a short segment of DNA which then binds the initiating nucleotide
- Can continue to transcribe RNA anywhere from few
bp to thousands of bp past the actual end of the gene - RNA is cleaved at a sequence-specific location and realised before transcription terminates
- Cleavage site occurs between an upstream AAUAAA sequence and a downstream GU- rich sequence
What happens to the mRNA to mature it?
- 5’ capping:
- Addition of 7-methylguanosine (m7G) to the 5’ end
- Protection from exonucleases
- Recognition for specific proteins of translational machinery
- Addition of polyA tail - plays role in stability and protection (don’t have = unstable and degraded)
Intron removal
- Carried out by protein/RNA complex called
- spilceosome
Alternative splicing - introns can be retained in the mRNA/exons can be spliced into the mRNA or skipped
What occurs after capping of mRNA
- Ribsosomes engage in the mRNAs and form a catalytic domain into which the tRNA enter with their attached amino acids.
- The proteins of the ribosomes catalyse all of the functions of polypeptide synthesis.
What are the rRNAs of the units of the ribsosome
- There are 4 major rRNAs in eukaryotic cells designated by their sedimentation size.
- The 28S, 5S, 5.8S RNAs are associated with large ribosomal subunit
- the 18S rRNA is associated with small ribosomal subunit
What do tRNA’s do in translation
- form covalent attachments to individual amino acids and recognise the encoded sequences of the mRNAs to allow correct insertion of amino acids into elongating polypeptide chain
What is required for transcription
- promoters
- RNA polymerase
- Transcription factors
- Enhancers
What do promoters do in transcription
- DNA sequences located upstream of the gene that direct the start site/direction
of transcription - consist of approx 40 base pair sequences that are located on the 5’ side of the transcription site and promote the ability of RNA polymerases to recognise the nucleotide at which initiation begins
- define start site for transcription and direction of transcription
why are transcription factors needed for transcription
- proteins that bind to promotor for it to function (signal)
What is the TATA sequence promoter?
- core promoter where RNA polymerase combines
- binds RNA polymerase and transcription factors to start process
- directs transcription
What is RNA polymerase and what does it do in transcription
- A multi-subunit protein
- RNA polymerase reads (copies) the gene (DNA) into messenger RNA.
- Similar to DNA polymerase in that it synthesises a chain of nucleotides using a template
- Recognises and binds to promoter sequences
- doesn’t need a RNA primer*
How many bases unwound in transcription bubble
- approx. 25 -> only 8 nucleotides will actually be paired (attached)
RNA polymerase error rate
1 every 10000 wrong -> lower because it can just destroy copy and make it again
What are the two domains of transcription factors
- Activatioon domain
- DNA-binding domain
what is the activation domain of transcription factors
- Binds to RNA polymerase and other proteins
- Assists formation of transcription complex on the promoter
- Affects DNA binding (more in LM)
what is the DNA binding domain of transcription factors
- binds to specific DNA sequence
- Consist of amino acids that recognise specific DNA bases near the start of transcription
- Transcription factors are typically classified according to the structure of their DNA binding
domains, which are generally one of the following types: zinc fingers, helix turn helix and
leucine zipper
How do transcription factors bind to DNA?
- Recognise” specific segments of DNA in the major and minor grooves and interact with them via hudrogen, ionic bonds or van der waals (hydrophobic) forces
- only recognise the outside of the DNA grooves/sites via structural motifs -> the geometry of the helix will be different for different sequences
- pattern of hydrogen bond donors and acceptors are only different in majoor groove and as a consequence most binding proteins bind in major groove
what motifs do transcription factors use to recognise DNA
Zinc fingers
Helix-turn-helix
Leucine zipper
What are zinc fingers
- Consists of specific spacings of cysteine and histidine residues that allow the protein to bind zinc atoms
- The metal atom coordinates the sequences around the cysteine and histidine residues into a fingerlike domain
- The finger domains can interdigitate into the major groove of the DNA helix and identify specific DNA sequences
explain helix-turn-helix
- Involved in protein dimerisation
- The motif is composed of two regions of a helix separated by a region of variable length, which forms a bend or turn between the 2 alpha helixes
- The recognition helix forms hydrogen bonds with bases in the major groove
Explain Leucine Zipper
- The leucine zipper contains 2 domains:
- the DNA binding domain (DBD) which contains basic amino acids and provides speciality
- Leucine zipper domain (LZ) which combines 2 polypeptide chains in correct shape for interacting with major grove. The leucine zipper domain is necessary for protein dimerisation.
- Leucine zipper proteins fingers fit into the major grooves of the DNA
What are enhancers and silencers
- Enhancers and silencers are regulatory sequences in the DNA which can either increase
(enhance) the expression of a gene (about 1000 fold) or reduce the rate of transcription initiation - DNA sequences that determine how frequently
and when transcription occurs - Bind specific transcriptional factors/activators
- Can be located long distances from promoters - Can act up more 50 kb away (though they can also be close)
- Can be upstream or downstream from the start site of transcription
Bi-directional: will function in either orientation
how does pituittary dwarfism occur?
- Occurs when mutations in LHX3 disrupt its ability to bind to promoter, so growth hormone is lacking
Clinical signs: - Underdeveloped kidneys leading to renal failure, - Under active thyroid - slow intelligence
- Alopecia is common
- Most dogs with pituitary dwarfism are sterile
- Treatment: Growth hormone replacement therapy (eg thyroxine)
How does Hemophilia B occur?
- Mutations affecting Factor IX promoter
- X linked recessive disorder
Clinical signs: - anemia and low platelet count
- Easily prone to haemorrhage from minor trauma
- Spontaneous bleeding
- Often occurs in joints and soft tissues
- Range of 4-6 spontaneous bleeds per year
- Treatment: blood transfusions and factor IX Activated factor IX activates
factor X - Cleaves prothrombin in active
thrombin - Converts soluble fibrinogen into insoluble fibrin
- Forms a fibrous mesh or clot that impedes the flow of blood
- Can result in uncontrollable and protracted (nasal) bleeding
how does T-cell Leukaemia occur?
- Over expression of c-Myc TF
- Levels determine the percentage of activated T cells that re-enter cell cycle
- Dogs clinical signs: anemia, thrombocytopenia (relatively few platelets in blood) and
neutropenia (presence of abnormally low number neutrophils WBC)
What is Myc gene amplication
- Essential TF, regulates proliferation, growth, apoptosis
- Myc gene amplification
- Most frequent cause of solid cancer
What is Androgen Insensitivity syndrome
- XY foetuses cannot respond to testosterone
- Mutations in androgen receptor (AR) gene
- Testosterone (T) diffuses from blood into cytoplasm of target cell
- Testosterone binds to the androgen receptor (AR)
- Complex binds to androgen response elements (AREs) on the promoter region of androgen- sensitive target genes in nucleus
- If AR doesn’t bnd testoosterone-target genes are not switched off
- XY individuals develop testis and produce testosterone, but they lack androgen receptors
because of a mutation - Cannot respond to hormones and develop feminine external genitalia but male internal
genitalia - Look and think of themselves as female
what are the components of AAs
- An amino group (basic)
- A carboxyl group (acidic)
- Alpha carbon (attached H atom)
- R group (variable)
what are the 4 levels of protein structure
Primary -> order of amino acids
secondary -> folding
tertiary -> 3D structure
quaternary -> monomer-monomer interactions
what is primary structure
- The primary structure of peptides and proteins refers to the linear number and order of amino acids present plus the location of any disulfide bonds
What is secondary structure
- Defines the steric relationship between amino acids that are close to each other in the primary amino acid sequence.
- Therefore, the ordered array of amino acids in a protein confer regular conformational forms upon the protein
- alphas helix and beta sheets + turns
what is an alpha helix
Right handed helix
- Carbonyl (C=O) of one amino acid is hydrogen bonded to the amino
- H (N-H) of an amino acid that is four down the chain
- R groups exterior
What are beta sheets
- AA’s adopt the conformation of a sheet of paper
- Anti-parallel sheets - PP backbone runs in opposite
direction - Parallel sheets - PP backbone runs in the same direction
What are beta turns
- Allow polypeptide chain to abruptly reverse its direction and connect antiparallel beta pleated sheets
- Only have quaternary structure if you have multiple polypeptide chains in a protein
What is tertiary structure
- Refers to the spatial arrangement of amino acid residues that are far apart in the linear amino acid sequence and to the pattern of disulfide bonds.
- Therefore, tertiary structure refers to the complete three dimensional structure of polypeptide units of a given protein.
- The tertiary structure of proteins may include, either mostly alpha-helixes, mostly beta sheets or a mixture of both connected by beta turns
What is quaternary structure
- many functional proteins contain 2 or more different polypeptide chains that are held in association by the same non-covalent forces that stabilize the tertiary structures of proteins.
- Proteins with multiple polypetide chains are termed oligomeric proteins.
The structure formed by monomer-monomer interaction in an oligomeric protein is known as quaternary structure. - Oligomeric proteins can be composed of multiple identical polypeptide chains or multiple distinct polypeptide chains.
- Proteins with identical subunits are termed homo-oligomers.
- Proteins containing several distinct polypeptide chains are termed heterooligomers.
what do tRNAs so in protein synthesis
tRNA molecules brings aa to mRNA - aminocyl-tRNA synthase attaches AA to tRNA
What is the wobble hypothesis
61 possible combinations of the 4 bases
- only 50 tRNAs
- weak interactions between third (wobble base) allows one
anticodon to associate with several different codons
What is initiation
- Large and small ribosomal unit, assemble onto mRNA
- Initiator tRNA (tRNAmet) enters P site
What are the sites of the ribosome and explain elongation
- P site: growing peptide residues
- A (acceptor) site: incoming tRNA attaches itself - Peptide bond is formed between the 2 AA’s
- E (ejector) site: exit site for tRNA after it has delivered AA’s
- The ribosome moves one codon further along the mRNA, releasing the empty tRNA
- A site is now free for the incoming tRNA
What is termination
-Ribosomes reaches a stop codon
- Recruits a release factor that signals for translation to stop
- PP is released and the
ribosome disassembles
- Signal sequence = proteins address
- Signal sequence at start = secretory pathway
- In middle = membrane protein
- Proteins must be transported to their appropriate destinations (in the cell or outside it)
Protein must be maintained in the translocation-competent state
- Must not misfiled or aggregate
- Specialised proteins called chaperones air in the folding proteins
What could happen if a protein doesn’t fold properly?
- Preferred: protein gets rapidly degraded - break back down into amino acids and reuse the amino acids
- Sometimes before a protein can be degraded, it will start to
aggregate (start to stick to itself) once aggregation starts, cell is no longer able to degrade it - More than 450 different disorders, which are known to affect all organs
how are incorrectly folded proteins degraded?
- Lysosomes OR
Ubiquitin marking: - Programmed destruction of proteins
- Addition of a chain of ubiquitin molecules of an internal lysine side chain of a target protein
- Directs protein to the proteasome
↳ Large multisubunit protease for degradation - Proteolysis of the ubiquitinated protein by a proteasome
what happens when mis-folded proteins aggregate
- During aggregation, destabilised proteins bind together
- Aggregates are highly stable
- Very difficult for cells to breakdown
- Continues to grow over time and may even recruit properly folded protein
- Often (but not always) toxic to cell
What causes proteins to misfold
- DNA mutations:
- Protein that cannot fold when synthesised
- Protein that is destabilised and unfolds easier once folded - Stresses during the lifetime of the protein modify it causing it to be destabilised and partially unfolded
- Environmental factors (metals, pesticides, toxins) can modify proteins making them more prone to misfold
- Post translational modifications can change
protein structure - proteolytic cleavage, sulfation, phosphorylation and ubiquitnation
What is Mad cow disease
- Transmissible spongiform
encephalopathies (TSEs) - Disease associated with accumulation of mis folded protein
- Infectious agent - small misfolded protein called prion
- Abnormal variants of naturally occurring
proteins (PrPc) in the brain (unknown function) - Infectious prions can cause correctly folded PrPc proteins to misfold and become prions (PrPSc)
- Domino effect: large numbers of misfolded prions
- Causes plaques which destroy the brain cells
- Inflammation and characteristic sponge like holes in the delicate membranes surrounding brain cells
How do folding issues cause cancer (p53)
- Disease in which impairment of folding results in reduction or absence of normal protein
- Suppresses abnormal cell proliferation
- Important mechanism for protein against cancer
- Acts in late G1 phase (prevents the cell progressing to the S phase)
- Involved in programmed cell death (apoptosis)
If p53 mutated - loses its ability to block abnormal cell growth
- Unable to fold properly and loses its ability to block abnormal cell growth
- These cancers are more aggressive, more apt to metastasise & more often fatal
how are mis folding diseases treated?
- Currently, no effective treatment but several options are being investigated.
For prion diseases:
- Reduction in PrPc expression - Inhibiting prion conversion - Stimulating the clearance of PrPsc
- Blocking PrPsc neurotoxicity
Other treatment options include chemical chaperones - enhance the folding and/or stability of proteins
