1
Q
what are the two purine nuleobases?
A
adenine and guanine
2
Q
what is the main structural difference between a single molecule of RNA and DNA?
A
the RNA has a hydroxyl (OH) group on the 2 prime carbon of the pentose sugar
3
Q
what are the 3 pyrimidine bases
A
cytosine, uracil and thymine
4
Q
what structural features do purines have?
A
2 linked cyclic structures, adenine has a NH2 group at the top of the larger ring and guanine has a =O in the same place
5
Q
what structure do pyrimidines have and what differentiates them?
A
single cyclic structure, cytosine has a NH2 at the top of its ring, uracil has a =O at the top of the ring, thymine has a =O at the top aswell as a CH3 group on the next carbon
6
Q
what is the central dogma of molecular biology
A
DNA transcription to RNA, translation to protein. (DNA replication and RNA to DNA reverse transcriptase also involved)
7
Q
where does transcription and translation occur? what are the two proceesses said to be seperated by?
A
transcription occurs in the nucleus, translation occurs in the cytosol (jelly like fluid that acts as a medium for organelle suspension). seperated temporally and spatially
8
Q
what direction does DNA replication occur in terms of nucleiotide sequnce?
A
5’ to 3’ (5 prime carbon to 3 prime)
9
Q
how do DNA nuliotides bind to eachother
A
via phosphodiester bond between the phosphate group 5’ and the 3’ carbon of the next nucleiotide
10
Q
how many hydrogen bonds do guanine and cytosine make aswell as adenine and thymine?
A
G/C bond is 3 hydrogen bonds between O–H,
N-H–N, N-H–0.
A/T has only 2 hydrogen bonds between N-H–O,
N–H-N.
11
Q
what is a nucleoside?
A
a base conjugated to a sugar. in NDA they are given the prefix ‘Deoxy’
12
Q
what is a nucleotide?
A
a nuclioside conjugated to one or more phosphroyl groups in DNA
13
Q
what are the names of nucleotides conected to a single phosphate group for G, C, A, T?
A
Deoxyguanylate (dGMP)
Deoxycytidylate (dCMP)
Deoxyadenylate (dAMP)
Deoxythymidylate (dTMP)
(can also replace ‘late’ suffix for ‘sine’ and add ‘monophosphate to the end to be more specific about the amount of phosphate molecules
14
Q
what physical features does DNA have?
A
DNA is comprised of two polynucleotide chains coiled around a common axis with a
right-handed screw.
Chains are anti-parallel (this is important later)
The “ideal” DNA helix has 10 base pairs per turn, with each aromatic base having a 3.4
Armstrong (Å) thickness partially stacked on each other, resulting in a helix pitch (rise
per turn) of 34 Å
DNA has equal number of adenine and thymine residues (A = T) and guanine and
cytosine residues (G = C) residues – known as Chargaff’s rules
15
Q
how do bases in the core of the double helix attract eachother?
A
van der waals forces
16
Q
what phase of cell division does DNA get replicated in?
A
S phase, arguably the most important as it must be error free to avoid mutation
17
Q
what features of DNA replication give it the title of ‘semiconservative’?
A
Each parental polynucleotide strand acts as a template
for the formation of the newly synthesised
complementary strand
This results in two molecules of double stranded DNA,
each consisting of one polynucleotide strand from the parent molecule and a newly synthesised
complementary strand (one strand from parent=semiconservitive)
18
Q
what is a replication bubble and its purpose?
A
the unwinding of two parental DNA strands exposing the bases, leading and lagging strand for replication.
19
Q
what is the name of a branch point of DNA where synthesis of DNA occurs?
A
replication fork
20
Q
how is base addition to newly replicated DNA strand determined?
A
hydrogen bonding with template strand
20
Q
what is the purpose of a RNA primer and where is it porduced?
A
|RNA primer gives DNA polymerase the 3’ hydroxyl group it needs to begin adding DNA bases in replication. RNA primers can be produced by DNA polymerase directly or the enzyme primase on the DNA strand
21
Q
what is the difference between the coding and template strand of DNA in replication?
A
The template strand (antisense) is the DNA strand read by RNA polymerase (3’ to 5’) to build complementary mRNA, while the coding strand (sense) has the same sequence as the resulting DNA/mRNA (RNA has T replaced by U) but isn’t directly transcribed
22
Q
what is DNA biosynthesis in terms of biochemical terms?
A
a biochemical reaction involving the hydrolysis of pyrophosphate (PPi) to two inorganic phosphate molecules (Pi), this is a reaction with a strongly negative delta G (exergonic/ spontaneous reaction that provides the energy needed for reaction from its reactants without need for outside energy input) driving reaction
23
Q
what does exergonic mean?
A
strong negative delta G, indicating a reaction is exergonic. releases energy and is spontaneous under standard conditions
24
what is the mechanism of two strand copying in DNA snd the differences between the two strands?
the leading strand is replicated continously as it is 5' to 3' direction, the lagging strand is antiparralel so is 3' to 5' making replication done in short fragments, process facilitated by Okazaki fragments (small DNA fragments that bind to the lagging strand and act as a end point for the polymerase to do a short replication towards) this leaves gaps which are sealed by DNA ligase enzyme
25
what are the 3 main types of RNA, the RNA polymerase that transcribes them and their purpose?
rRNA (ribosomal RNA) transcribed from rRNA-encoded genes 47S as a template for ribosomes and is transcribed by RNA polymerase 1
mRNA (messanger RNA) us transcribed from protein encoding genes as a template for the synthesis of protein, transcribed by RNA polymerase 2
tRNA (transfer RNA) is trancribed from tRNA encoding genes and is essential for translation as it carries activated amino acids to the ribosomes. is transcribed by RNA polymerase 3
26
what are the 3 steps of mRNA synthesis by RNA polymerase 2?
initiation: RNA Polymerase II and transcription factors assemble at promoter sites and unwind the DNA so that
RNA synthesis can then be initiated
Elongation: RNA polymerase II moves along the DNA template to synthesise the newly produced mRNA in a 5’ to 3’ direction.
The sequence of growing mRNA is driven by base-pairing to the template DNA strand, faithfully copying the DNA, therefore an accurate copy of the DNA template is produced
Termination: RNA Polymerase II ceases transcription at a defined site determined by the termination sequence
The mRNA transcript is then stabilised by specific modifications
27
what are 2 promoters used in transcription start site, what do they do and what important features do they have?
the CAAT box
the TATA box/hogness box
they recruit RNA plymerase 2 and define transcription initiation.
they can be on the template (antisense) or coding (sense) strand and can contain a GC box (GGCGGG)
they are located upstream from the start of RNA initiation usually less then 100 bases upstream
28
how is initiation of mRNA creation tightly regulated?
by many proteins which are collectively called transcription factors (TF)
TF Recognise specific DNA sequences near promoters of genes
TF have characteristic DNA-binding structures
TF are targets for signalling pathways
Can be activated by phosphorylation and subsequent movement into the nucleus
29
what are enhancers and their role in transcription
found kilobases upstream of promoters. they bind transcription factors and can be negative or positive regulators either turning genes off or on respectivly
30
what and when does RNA transcription elongation occur?
Following transcription initiation, mRNA is synthesised by RNA Polymerase II during a process called transcription
elongation. In this phase, the template DNA strand is copied to produce a complementary mRNA strand, forming a
transient RNA-DNA hybrid helix. The resulting mRNA sequence is nearly identical to the coding strand, except that
uracil replaces thymine.
31
what happens in transcription termination
RNA Polymerase II and
nascent mRNA are released from the DNA template strand
Timely transcription termination is essential for genome expression and integrity
Occurs at defined sites and is regulated by helicase Sen1
mRNA is very unstable, so it is processed in two ways:
-A modified 5’ cap
-Poly(A) tail at the 3’ end which are both Needed for stability and helps with translation
32
what two modifications are made to pre-mRNA to improve its stability?
a modified 5' cap end is added
a poly A (Adenine) tail at the 3' end
33
when does 5'-capping occur and what does it involve?
occurs when pre-mRNA is around 25 nucliotides long
involves specific capping proteins and a rare 5'-to-5' triphosphate bridge with GTP (guanisine triphosphate)
34
what is an endonuclease?
enzyme that cleaves DNA/RNA
35
what motif signals for mRNA cleavage and poly(A) tail binding?
5'-AAUAAA3'
36
what does the polyadenylation at the 3' end of mRNA do and how long is the polyA tail?
protects mRNA from degredation and enhances ribosome recriutment to mRNA aswell as assisting in translation initation.
>250 nucleiotides of raw A
37
what are introns?
intervening DNA/ non-coding regions. their size can range from 50 to 10,000 nucliotides
38
who discovered mRNA splicing?
Richard J roberts and Philip A sharp independantly in the late 1970s
39
what is the correlation between number and length of introns and complexity of organism?
the more complex the organism, the lunger and more HIGH NUMBER the introns are
40
what purpose does introns serve?
alternative splicing
gene expression
evolution
protein diversity
mRNA transport
Chromatin assembly
nonsense mediated decay
buffering
41
how does intron splicing occur?
intron bunches up like pushing a section of rope from both sides, intron cut out and the now close together exons either side of the excised intron are joined together
an excised intron is known as a lariat
42
what do all introns begin and end with?
begin with GU and end with AG
43
what is key to intron splicing?
pyrimidine (C,T, U) rich tract and specific adenine in the intron key to its splicing with the A nucliotide being known as the branch site
44
what are snRNAs and what do they stand for?
small nuclear RNAs which have less then 300 nucliotides and catalyse the splicing of pre-mRNA.
they form snRNPs with specific proteins, 'small nuclear ribonucleoproteins' which recruit other snRNPs into a large complex called a splicosome which can capture, splice and release RNA acuratly
45
how do snRNPs act on introns?
they bind to the intron and cause it to fold bringing the 5' and 3' ends closer together making a loop, this pulls the exons either side together so when the intron detaches as a larait they join together in mature mRNA
46
what is constitutive splicing
the removal of introns and joining of al exons from a gene in a fixed order to produce ONE type of mature mRNA.
only around 5% of genes undergo constitutive splicing
47
what is alternative splicing
different mature mRNA strands generated from the same initial transcript of a gene, it can generate proteins with completely different functions, a powerful mechanism for protein diversity as well as regulating gene expression and function
48
for a gene containing 6 exons and 5 introns, how many different mRNAs can be generated?
16 including the full length protein containing all 6 exons
49
how is alt splicing controled
by binding of trans acting splicing factors to cis acting elements in pre-mRNA, the cis elements include sequnces that help recognise exons and modulate splicing
50
what are SR proteins
found in nuclear granules and can interact w RNA pol 2.
signaling events can modulate the phosporylation status and alt splicing of SR proteins can change their expression patterns and function
51
what are nuclear speckles
regions in nucleusthat contain high conc of splicing factors usually found nearby genes that need splicing
52
what are the two types of RNA splicing control?
negative (repressor prevents splicing of introns)
positive (activator signals for the cutting out of introns)
53
why is RNA goated?
it can act as an enzyme and splice itself, and is a form of genetic storage.
54
ow does RNA self splicing occur?
short stretches of RNA nucleotides bond with other regions to create folds that drive splicing within structure
55
what are tRNAs called when they bind to their specific amino acid?
aminoacyl-tRNAs
56
what 3 letter codon do all protein synthesis begin with?
AUG (adenine-uracil-guanine) which signals for methionine
57
what 3 codons signal for protein synthesis termination?
UAA, UAG, UGA are the 3 terminal stop codons
58
what is the UTR?
untranslated region, it is a area near the 5' end of the protein that dosnt affect sequence of amino acids in a protein, so its FUCKIN USELESSSSSS!!!!!!
59
how do tRNAs bind amino acids
by aminoacyl-tRNA synthetases whichbind both the tRNA and amino acid and bind them together before releasing them
60
what are the 3 binding sites of the ribosome?
amino acid site (A) where aminoacyl tRNA anticodon pairs up with mRNA codon ensuring correct amino acids is added to polypeptide chain (synthesised protein)
polypeptide (P) site where the amino acid is transfered from tRNA to polypeptide chain
exit (E) site where empty tRNAs sit before being released back to the cytoplasm to bind another amino acid.
61
what 3 stages make up translation and what occurs during them?
initation: initiation tRNA holding methionine (met-tRNA) scans mRNA for the AUG start codon in a step requiring energy. when it finds AUG, the large ribosomal subunit and elongation factors are recruited to form the 80S initiation complex, officially begining synthesis.
elongation: riosome moves across mRNA from 5' to 3' in a process known as translocation requiring translation factor G. the tRNA after the start codon methionine tRNA binds to A site, requiring GTP as energy source. peptide bonds between first and second amino acids are formed through peptidyl transferase activity, ribosome shifts casing empty tRNA to ocupy the E site freeing up room for the next tRNA to bind and release its amino acid, empty tRNA released, cycle continues
termination: one of the three stop codons (UAA, UAG, UGA) signal the end of translation, tRNAs dont recognise this codon as none have a anticodon matching it. instead release factors (RF) bind to stop codon at the A site triggering cleavage/release of polypeptide from robosome and ribosome is disassembled with the help of RF
62
what do we mean when we say genes are expressed?
when a gene has been transcribed into mRNA creating a protein
63
what role do epigenetics play in determining which genes are expressed?
Modifications to the genome that affect gene expression but do not alter the DNA sequence
Epigenetic changes are heritable alterations of gene expression and chromatin organisation
Can be reversible unlike DNA changes – thus can be modulated – more to come in lecture 4
They add an additional level of gene expression regulation to the cells.
64
what is the main modification brought by epigenetics?
The main epigenetic modifications consist of DNA methylation
65
what is DNA methylation?
adition of methyl groups to nitrogen bases of nucliotides, mianly cytosine sometimes adenine. it influnces the structure of DNA regulating gene expression by recruiting proteins involved in gene represion or by blocking binding of transcription factors to DNA
66
what is CpG?
a rare dinucliotide sequence, more rare then statisticly should be showing intent/ directed evolution. 5'-CpG-3' with the p representing the phosphate residue in the DNA backbone, easily methylated, methyl groups protrude into the major groove of DNA interfering with TF binding
67
what are CpG islands?
regions of the genome high in CpGs, enriched in promoter regions of genes
68
what role do CpG islands have in promoters function?
they undergo chemical modification which alters binding of TFs
69
how does CpG methylation actually work
cytosine is methylated in CpG by DNA methyltransferase (DNMT)
DNMT1 actively maintains DNA methylation during cell division ensuring daughter cells retain modification, making it a heritable trait.
70
what is maintenance methylation?
ensures methylation patterns persist in daughter cells
71
what is de novo methylation?
a process allowing different methylation patterns during development
72
what role does demethylation play in the human body?
reactivates silenced genes or incorrectly methylated bases as DNA methylation can be altered as a result of small nuclear polymorphisms, diet and enviromental factors
73
74
how is DNA organised?
packing proteins like histones pack DNA into smaller bundles, the DNA-histone octamer complex is called a nucleosome while the overall DNA-histone complex is called a chromatin
75
how does histone work?
four subunits of histone assemble into a octamer which DNA wraps around, this is called a nucleosome while on a larger scale it is refered to as a chromatin
76
what is histone acetylation?
acetylation is universaly associated with gene activity, acetylation of histones neutrilises positive lysines, decreasing the attraction between the positive histone and negative charged DNA, loosening its form from a hetrochromatin to a euchromatin
76
what are the two chromatin states?
Euchromatin:
Loosely packed form
Enriched in genes
Under active transcript
Heterochromatin:
Tightly packed form of chromatin
Generally genetically inactive
Inaccessible to RNA polymerase
77
what acetylates and deacetylates histones?
Histones are acetylated by histone acetyltransferases (HATs) and
deacetylated by histone deacetylases (HDACs)
78
how does methylation interact with histones?
can either increase transcriptional activity or silence transcription, histone methylation is regulated by Histone methyltransferases (HMTs): which Catalyse the transfer of methyl groups from S-adenosylmethionine (SAM) to lysine or arginine
residues on histones, and Histone demethylases which
Remove the methyl groups attached to lysine residues.
79
how is epigenetics and histone changes linked to cancer and disease?
phosphorylation of a tumour supression gene can inhibit gene expression leading to a lack of tumour supressents
genes can be turned on by changing histone modification pattern
80
what is a organism that uses epigenetics extremely?
bees, the difference between a queen and worker bees are what DNA bases are methylated, the reason why royal jelly can turn a bee into a queen is because it contains compounds that inhibit methyl transferase, triggering DNA demethylation in bee larvae. they eat the honey that makes them slay.
81
what is RNA interference?
dsRNA (double stranded RNA) is injected into a cell or organism causing it to knockdown specific protein production, this is done by dsRNA blocking translation of mRNA with sequences similar to the dsDNA. applicable if you wish to knockdown specific protein production.
requires small interfering RNAs (siRNAs)
82
what are microRNAs and their purpose
miRNAs modify gene expression by binding to mRNAs and interfering with their translation or promoting their degredation in a process called 'post-transcriptional gene silencing' they can even target epigenetic factors like DNA methyltransferase which can increase gene expression.
83
what is CRISPR?
a speciific guide RNA (gRNA) is synthesised that directs endonuclease enzyme Cas9 ro specific DNA target sequence, this can cleave DNA at a precise point allowing insertion or deletion of nucliotides
84
why do cells signal?
to co-ordinate day to day physiology and regulate behaviors in multicellular organisms, at a cellular level its reuired for the cell to rspond and change to its enviroment, failure and impairment leads to disorder, disease and death.
85
what physiological activites are regulated by signaling of cells?
metabolism
cell growth
devision
differentiation and development
gene expression
motility (think chemokines)
morpholigy
death
BASICALLY REMMEMBER EVERY SIGNALING MOLECULE FROM BM212
86
wht are the two mechanisms of cell communication?
intercellular signaling: between cells, permits single cell to influnce behavior of other cells in a specific way
intracellular signaling: within the cell in response to extracellular and intracellular stimuli, e.g second messanger generation
87
what are the 4 types of intercellular signalling?
-autocrine, where a cell targets itself, e.g inflamatory cell secreting IL2 which influnces itself
-paracrine, where a cell targets a nearby cell
-juxtacrine, where a cell targets a cell connected to it by gap junctions (think nerve cells and synapses)
-endocrine, a cell targets a distant cell through the bloodstream, often by hormones
88
what 2 molecules are often involved in intracellular signaling and their function?
secondary messangers, which amplify and relay extracellular signals to targets within the cell, vital in signaling with molecules that cannot cross the cell membrane
protein kinases, which act as on/off switches by phosphorylating specific molecules
89
what are the 4 major types of cell signaling receptor and the timescale they opperate at?
-ligand gated ion channel receptors, millisecond
-G-protein coupled receptors, seconds
-kinase linked receptors, hours
-nuclear receptors, hours/days
90
how are GPCRs influenced by different signals?
a single gpcr can be influnced by multiple signals, which can alter the what, where, and when parameters of G-protein-coupled-receptors, ultimatly effecting the kinectis of the response
91
what are signalosomes?
Large supramolecular complexes Composed of unique combinations of signalling pathway
components targeted to discreet intracellular localisation via the
association of anchor or adaptor proteins. Signalosomes are very adaptive and dynamic, Allowing cells to construct the optimum cellular subdomain for
signalling
G proteins and effectors already colocalised (meaning their fate and location are already determined, thus uneffected by signalosome's adaptive nature
92
what signals contribute to gene expression determination?
nutrients like carbohydrates, fats, amino acids.
the link betwen nutrient intake and gene transcription and epigenetic regulation is strong
93
what link does S-Adenosylmethionine (SAM) have with epigenetic regulation? example :)
SAM is a methyl doner required for DNA methylation synthesised in the methionine cycle from several diet precursors: methionine, folate, choline, betaine, vitamins B2, B6, B12. a lack of these precursors results in low SAM synthesis and DNA hypomethylation.
FOR EXAMPLE IF YOU LACK FOLATE WHILE BABY GESTATION DNA HYPOMETHYLATION OCCUR RESULTING IN disabilities through nural tube defects (already knew of that but now we know why a lack of folate causes nural tubes to not close)
94
how do cancer cells evade the immune system using signaling pathways?
Activation of ligand-activated receptors lead
to increased gene transcription of
immunoregulatory molecules such as the
checkpoint programmed death-1 (PD-
1)/programmed cell death ligands (PD-Ls)
promote immune evasion of tumour cells.
95
what is a common signaling mediator that can influnce many cell types and what receptors do these cells posses?
Acetylcholine which binds with heart pacemaker cell's muscarinic 2 (M2) acetylcholine receptor (GPCR) which couples to Gi to decrease cAMP in the cell, salivary gland cells which express M1 and M3 receptors which couple to Gq to upregulate PLC and intracellular calcium and also acts on nicotinic acetylcholine (non-selective cation channel) receptor present on skeletal muscle (no purpose listed!!)
96
how do cells turn off signalling?
phosphodiesterases (PDE) catalyse the breakdown of cAMP, also able to create discreet cAMP gradients in cytosol
97
what process can be used to measure cell signaling?
immunoblotting (gel electrophoresis, western blotting) and ELISA which isnt immunoblotting
97
what is the link between signalling pathway activation and DNA mutation?
no direct link but their dysregulation can cause conditions that then increase probibility of mutations arising and persisting
98
what are 3 examples of signalling pathways that lead to cancer when dysregulated?
RAS/MAPK Pathway:
Mechanism: Oncogenic mutations in RAS (e.g., KRAS^G12D^) hyperactivate MAPK signalling → uncontrolled proliferation + replication stress
Result: Cells accumulate DNA replication errors and double-strand breaks
Example: Seen in many pancreatic and colorectal cancers, where KRAS mutations drive further genomic instability
PI3K/AKT Pathway:
Mechanism: Hyperactive PI3K/AKT signalling promotes survival even in the presence of DNA damage. It also downregulates pro-apoptotic proteins
Result: Damaged cells that should die instead survive and replicate with mutations
Example: Breast cancers with PIK3CA mutations often show increased genomic instability
p53 Pathway:
Mechanism: Normally, p53 halts the cell cycle for DNA repair or induces apoptosis. If signalling through p53 is disrupted (e.g., by mutations in MDM2 or upstream pathways), damaged DNA isn’t repaired
Result: Mutations accumulate unchecked
Example: Li-Fraumeni syndrome, where defective p53 signalling predisposes to multiple cancers due to impaired DNA damage response
99
what are the 3 types of DNA mutations?
-point mutations: change within one single DNA base (A->G)
-frameshift mutations: an insertion or deletion of nucliotide bases that isnt in multiples of 3, making all codons shift changing the entire reading frame of the genetic code (DEADLY)
-chromosomal mutations: changes to structure or number of chromosomes, large scale changes that have significent effects on gene expression (split into 5 types)
100
what are the 5 types of chromosome structure mutation?
-Deletion: Loss of a chromosome segment.
-Duplication: A segment is copied, creating extra genetic material.
-Inversion: A segment breaks, flips, and reinserts backward.
-Translocation: A segment moves to a non-homologous chromosome.
-Insertion: A segment is added to a chromosome (sometimes grouped with duplication/translocation)
101
what are the 3 types of point mutations?
insertion, extra base added (frameshift mutation if not adding 3)
substitution, one base replaced by another (generally least harmful but if codon changes to amino acid of different type or a stop codon then the protein made will certainly not function)
deletion, a base is removed (frameshift if not done in 3s)
102
what are the 3 consequences of point mutations
silent mutation: leaves protein unchanged (some codons code for the same amino acid so swapping from GTA to GTT will cause Val amino acid to still be produced leaving no change to protein)
-missense mutation: alers single amino acid (slightly harmful but as long as it isnt too different from structure of unmutated amino acid coding codon should be minimal)
-nonsense mutation: creates a premature stop codon (welp, you made a UAA, UAG or UGA and now the protein is spat out half finished, hope that protein wasn't important cuz it certainly isn't anymore)
103
what is an example of disease caused by point mutation?
NIemann-pick disease, an inherited lysosomal storage disorder where the body cant properly break down and transport lipids, lipids accumulate in organs leading to 'enlarged' organs (having a big brain in this case is not good) this is caused by a point mutation in a protein (enzyme) that breaks down and transports lipids
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