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Lecture notes, lectures 12-23 - DNA lectures

Biochemistry And Molecular Biology (University of Melbourne)

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DNA Summaries

Lecture 12:
 1950s – knew genes were inherited via chromosomes but unsure whether protein or DNA
Proteins DNA
Amino acids in sequence (20 letters)  variation 4 letters in sequence (ATGC)
Protein structures varied DNA looks similar
Enzymes studies
Proteins unstable DNA stable
 Erwin Chargaff:
o DNA-based content different b/w species
o DNA the same within one organism
o Does not vary over time/environment (stable)
o A=T
o G=C
 Rosalind Franklin, Maurice Wilkins X-ray crystallography of DNA indicated double helix and periodicity
 Watson and Crick developed double helix DNA model
o Satisfied periodicity and equal A-T, G-C ratio
o Satisfied replication and translation – one strand acts as a template
 Transcription of DNA into complementary RNA
 Translation of RNA on ribosome into polypeptide chain
 RNA
o Nucleic acid
 Different types of base pairing with modified bases
 E.g. Triple base pair: cytosine+guanine+7-methylguanine  distortion of duplex
o Double stranded in sections, more flexible  loops + varied 2o structure
 Hairpins – exact reverse complement in a single strand
 Internal loops (bulging)
o Involved in translation and transcription
o Makes riboprotein complexes which carries out enzymatic functions
o Regulates gene expression
o Viruses  RNA = genetic material (retrovirus = reverse
translation, RNA  DNA)
 Nucleotides
o Make up nucleic acids
o Phosphate + pentose sugar +(N-β-glycosidic bond)+ base
o Pentose
 Ribose = RNA
 Deoxyribose = DNA (C2 no OH)
 Linear = aldehyde
 Circular = β-furanose
o Bases
 Adenine, Guanine = purines = two rings
 5C ring not flat  puckered
 4 structures depending on
whether C2 or C3 up/down
 Cytosine, Thymine/Uracil = pyrimidines
 A-T = two H bonds

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 G-C = three H bonds (stronger)

 DNA single strand has 5’ and 3’ end
 Covalent phosphodiester linkage b/w phosphate and 3’ on neighbouring
pentose
 DNA double stranded and antiparallel (complementary)
 Double helix has major groove + minor groove, with phosphate backbone
exposed to outside and bases inside
 B DNA (Watson-Crick model), A form (laboratory conditions – shorter distance
b/w adjacent grooves), Z form (left handed helix)
o RNA-RNA double helix takes A form due to conformational constraints of C2 hydroxyl group

Lecture 13:
 DNA replication occurs during interphase
o M phase is when chromosomes divide
 Proteins needed so that nucleotides can be connected to template strand with phosphodiester bonds
o New nucleoside triphosphate added with 2Pi cleaved

DNA polymerase – 5 types in E.coli (2 involved in DNA replication and others in DNA
repair)
 DNA polymerase I – functions in repair and recombination
o Wraps around DNA in pocket with palm, thumb and fingers
o Adds nucleotides along DNA template strand 5’ 3’ processive
o 2nd active site for exonuclease activity
 Polymerase repositions the mispaired 3’ terminus (nucleotide)
into the 3’5’ exonuclease site
 The newly vacant 3’ terminus repositions back to the polymerase active site, adds
correct nucleotide
 Excised nucleotide with one phosphate goes back into pool since only triphosphates can
be added to strand
 DNA polymerase III – main polymerase in DNA replication (we need 2, one for each strand)
o Much bigger complex with more proteins
o 2 β sliding clamps keep DNA associated with polymerase
o Can only operate on 3’ end
o Adds nucleotides onto 3’ end of new strand; strand grows 5’3’
o Asp on DNA Poly II co-ordinates with 2xMg2+ which co-ordinates with the phosphate oxygen
already on growing strand and the phosphate oxygen on nucleotide to be inserted
o RNA primer needed to start double stand on growing strand of DNA
 RNA does not need double strand to start synthesis
 DNA replication makes 1 mistake every 109-1010 nucleotides (1/1,000-10,000 replications in E.Coli)
o Binding of nucleotides and proofreading ensures accuracy
o Incorrect base pairing doesn’t fit in enzyme pocket
DNA Polymerase I DNA Polymerase II
Subunits 1 >10
Mr 100,000
3’5’ exonuclease (proofreading, Yes Yes
removal of nucleotide backwards)
5’3’ exonuclease (i.e. removes in Yes No
forwards direction)

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Processivity 3-200 >500,000

Initiation of Replication:
 Prokaryotes – single replication origin with replication forks in either direction
 Eukaryotes – multiple replication bubbles with replication forks in either direction which eventually join
 more efficient for more genetic material
 DnaA proteins recognise and bind to the origin sequence (oriC, 4x9bp) - Ecoli
o Forces DNA into tighter sequence, higher tension  overwinding
o Active DnaA requires ATP but does not hydrolyse it
o DUE sequence (rich in A=T pairs, 3x13bp) opens/denatures by itself under tension
 DnaB protein (helicase) loads onto open DUE sequence and unwinds DNA
o 6 subunits which spin as it unwinds DNA and separates base pairs, using ATP
 DnaG protein (DNA primase) synthesises RNA primers
 Single-stranded DNA-binding protein (SSB) binds and protects single-stranded DNA
 DNA gyrase (DNA topoisomerase II) relives torsional strain (by forming isomers) generated by DNA
unwinding by underwinding DNA just ahead of replication fork
 Semiconservative

Leading/Lagging Strand:
 Leading strand adds nucleotides continuously towards replication fork
 Lagging strand has Okazaki fragments (5’3’) added away from fork for about 1000 base pairs
o Lagging strand looped around so it can be replicated in correct direction at same time as leading
strand
o DNA polymerase III detaches when it reaches next RNA primer (and previously synthesised
fragment) and moves up towards fork to make new fragment
 β clamp releases lagging strand
o Clamp loader swivels around and attaches the β clamp and transfers this to DNA Pol III, creating
a new loop which grows as nucleotides added
 DNA polymerase 1 removes RNA primer (since it has exonuclease activity 5’3’ direction) and
replaces RNA with DNA one nucleotide at a time
 DNA ligase forms phosphodiester bond b/w DNA, joins 3’5’ direction

Telomeres:
 Primer added to end of linear chromosomes, gaps when removed

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 Telomerase adds new DNA at the ends so they are not degraded and lost with each replication
o Telomerase is a reverse transcriptase that carries a short stretch of RNA which hybridises to
parent strand and allows its extension

Lecture 14: DNA repair

 Cells suffer 60,000 DNA lesions in a day  radiation, chemicals, cell metabolism with oxidising agents
 Mutation – change in DNA sequence, passed onto next generation if occurs in gonads  cancer risk
 DNA polymerase cannot move past lesion during replication  entire rest of DNA strand
lost/unreplicated
o Cell damage blocks cell cycle progression  checkpoints prevent cell from entering next stage
with lesion
o Lesions need to be repaired before S phase (DNA replication) and before mitosis
 Programmed cell death (apoptosis) when cell cannot progress through cell cycle or unable to function
o Cell death adjusts number of nerve cells to the size of target  refines neural connections
o Self-recognising B cells also destroyed to prevent autoimmune problems
 Cancer cells avoid apoptosis, mutation disrupts DNA repair or cell cycle checkpoint  mutations
accumulate

Mismatch Repair:
 Specific to E.coli
 Error during replication that was not corrected by the polymerase
 11x GATC sequence (palindrome on other strand, located in OriC locus) has A methylated by Dam
methylase
o NB: Not the same as chromatin remodelling in eukaryotes – cytosine methylation
o Hemi-methylated state for short period where proteins can distinguish b/w old and new
strand and identify mismatch
 Dam methylase adds CH3 other strand after a few minutes so that DnaA binding occurs for replication
 Nick in new strand near the mismatch, exonuclease (e.g. helicase) gets rid of short sequence of bases
and DNA Pol III fixes region
o Reasonably expensive mechanism, uses ATP to take out bases  important process

Single Strand Base Excision Repair:

 Damage to single base:
o Deamination = removal of an amine (NH2) group due to water (hydrolytic attack)
o Cytosine deaminated to uracil
 DNA proteins know uracil should be cytosine since uracil only in RNA
o 5-methylcytosine deaminated to thymine
 Harder to identify this mismatch
o Adenine  hypoxanthine, guanine xanthine
 Depurination (more common), depyrimidination  base comes off pentose backbone
 Spontaneous oxidation and uncontrolled methylation
 Repair:
o When U instead of C, uracil glycosylase removes U  base-sized gap in double strand
 Gap also represents depurination issue, same steps follow
 NB: 4 DNA glycolyases for uracil in humans
o AP endonuclease cleaves phosphate backbone next to where purine has been removed
o DNA polymerase I uses 5’3’ activity to remove sequence of bases and put new ones in
o DNA ligase repairs nick

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Nucleotide Excision Repair: for larger lesion

 Thymine dimers occur when covalent bonds form with neighbouring purines instead of base-pairing
o Cyclobutane thymine dimers have 2 parallel covalent bonds
o 6-4 photoproduct has diagonal covalent bond
 Causes DNA kink which makes DNA inaccessible but easy for cell to identify problem
 Exonuclease cleaves/nicks phosphate backbone in DNA a small region away from lesion
 DNA helicase unwinds section of double helix, 12bp section (prokaryotes, 30bp = eukaryotes) and
lesion comes off due to tension
o Helicase needed to access large lesion, since DNA Pol I only repairs one nucleotide
 DNA Pol I repairs DNA
 DNA ligase repairs nick

Double Strand Break Repair:

 Caused by ionising radiation, DNA replication errors, oxidising agents
 Backbone damaged on both strands  held in place by histones
 Susceptible to virus attack, ends can degrade chemically
 Most repaired within 24hrs but 25% of repairs contain errors  mutation
 Non-homologous end joining
o Loss of nucleotides due to degradation from ends
o End joining but some deletion of DNA sequence
o Usually not problematic since genes only 1.5% of DNA but deletions can accumulate

Lecture 15: Transcription

 DNA  mRNA
 RNAs encoded by genes which are only produced by transcription (not translated)
 Only one strand involved in transcription, synthesised from the template strand in a 17bp bubble
o Template and new transcript are reverse complement of each other (not identical)
o Coding strand almost identical to RNA transcript but U instead of T
 RNA polymerase has 4 tunnels for DNA in/out and RNA in/out
o RNA nucleotides (ribose) enter
o Whole enzyme complex covers 35bp but only 17bp open
o Asp residues co-ordinate Mg2+ which interact with phosphates on incoming NTPs
o β, β’, 2xα, ω and σ70 (sigma) subunits
 Sigma subunit binds -10 to -35 and helps separate strands, leaves complex when
assembled and replication starts

 Template strand the bottom strand

o Non-template strand (top strand) also called coding strand

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o DNA always has 5’ to the top left, promoter is left of the 5’ of coding strand
 RNA peels off as it is made 5’3’
 Different genes may be transcribed from different strands
 RNA promoters
o Upstream=negative from RNA start, downstream=positive from RNA start
o Transcription machinery recognises -35 region, -10 region upstream

Eukaryotes:
 RNA polymerase I – encodes pre-ribosomal RNA which is processed
 RNA polymerase II
o 12 subunits
o Conserved structure, function and mechanism  similar to E.coli RNA polymerases but more
complex
o Transcribes/expresses almost all genes that encode mRNA to be translated into proteins
o Recognition sequence for RNA polymerase = TATA box (= TATAAAAA = directional)
o Requires transcription factors
 RNA polymerase III – tRNAs and splicing RNAs

Transcription Factors:
 General transcription factors  required for expression of almost all genes
o TBP = TATA Box binding Protein
 First protein that recognises promoter sequence
 Bends the DNA at the promoter (TATA box)
 Easier to open? Or easier for region to be recognised by other proteins?
o TFIIB binds to TBP, recruits RNA Pol II to bind to TATA box
o TFIIH has helicase activity, unwinds DNA at promoter so transcription can start
 Not the same helicase (DNA replication) but similar activity in unwinding
 TFIIH phosphorylates CTD (C-terminal domain) of RNA Pol II which alters the structure so that RNA
polymerase can transcribe
o CTD domain has repetitive structure allowing it to coil in β spirals, very flexible  different
shapes bind to different proteins (like regulation centre for RNA Pol II)

 Gene regulatory proteins (transcription factors)  regulate expression of a subset of genes

o Elongation factors
o Termination factors – we lose phosphorylation of the CTD

mRNA Processing:
 After transcription, pre-mRNA (primary transcript) has to be processed to make it more stable
o Markers regulate RNA’s stability since it is unstable and can be degraded
o Processing occurs while mRNA still being produced
 CTD controls initiation elongation, termination, 5’ cap, spliceosome and polyA tail
 5’ cap
o Modified nucleotide (7-methylguanosine) connected in completely different way, signal stability
 2x 5’ joined together instead of 5’3’
 All 3 phosphates of nucleotide are in between two nucleotides
o Cap synthesising enzyme on CTD
 As RNA comes off, cap is put on and tethered to the CTD (controlled by CTD)
 Splicing introns

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o Eukaryotic genes have introns that need to be spliced to form mature RNA
o Spliceosome (proteins + RNA composition) subunits called snRNPs (U1-6 = forms of snRNP)
o Recognisable sequences in the intron
 GU at the start - U1 snRNP base pairs to ψ (modified bases) located after GU
 A in the middle
 U2 binds to A and sequence around it
 Rest of the proteins (U4-6) bind and sequence loops up, bringing A over to GU
which bind and a cut is made precisely at end of LHS intron
 AG at the end, which is then cut and two ends are spliced together with no errors
 Lasso of DNA released (Lariat structure)
 ATP used for this process
o Splicing occurs on CTD  snRNPs bind to CTD
 As RNA produced, first intron binds to spliceosome and spliced out during transcription
 Poly A tail
o Preserves RNA
o At the end of 3’ end of eukaryotic mRNAs there is a sequence AAUAAA but transcription
continues
 An exonuclease cleaves the mRNA after its AAUAAA sequence (attached to the CTD)
 This sequence recognised by polyadenylate polymerase which adds many A’s after
o Determines how long a protein hangs around: long tail = long time

Lecture 16: Translation

 Translation in cytoplasm
 Ribosome reads of the genetic code in codons (3 nucleotides = 1 codon)
 Degenerate codons; multiple codons for one amino acid
o Wobble in the third base of a codon (=1st base of anticodon) – only recognised in U, C, A
o One tRNA can read more than one codon
o Inosine (altered base) can base pair to multiple nucleotides (A, U, C)
 Other altered bases = pseudouridine (splicing RNAs), 7-methylguanosine, 4-thiouridine
o 64 codons  61 = amino acids, 3 = stop codons
o 20 tRNAs; 1 for each amino acid
 Non-overlapping genes, no space b/w genes
 Punctuations (start + stop codons)
o Start: AUG, also codes for fMet
 Every single protein starts with methionine, which can be cleaved later
o Stop: UGA, UAA, UAG (you go away, you are away, you are gone)

Aminoacylation
 Aminoacyl-tRNA covalently attaches amino acid to tRNA using ATP
o Carboxyl end of amino acid has ester linkage with 3’ end of tRNA
o Can have monomeric and dimeric enzyme
o Dimer attaches amino acids to 2 tRNAs at once
o 20 aminoacyl tRNA synthetases for each amino acid and all its correct
tRNAs
 Amino acid arm with amino acid language, 3’ end
 Anticodon arm (with nucleic acid language, 5’ end) binds to codon on mRNA

Mutations:

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 Nucleotide-pair substitution effects:

o Silent: if mutation has same amino acid encoded
o Missense: different amino acid put in place but cannot tell whether function altered
o Nonsense: premature stop codon, protein truncated, amino acids missing, changes function
 Nucleotide-pair insertion/deletion effects:
o Frameshift: causing immediate nonsense (1 nucleotide-pair insertion)
o Frameshift: causing extensive missense (1 nucleotide-pair deletion)
o No frameshift but one amino acid missing if 3 nucleotide-pair deletion, everything else
downstream of deletion is okay
 Start codon AUG dictates where reading frame starts
 Protein effects:
o Deletion of a section
o Bent into incorrect shape
o 1 amino acid in catalytic triad replaced  no enzyme function
o Can’t bind metal ion essential for function
o Can no longer bind to cofactor
o Can no longer bind to another protein
o Can no longer bind to DNA

Initiation, Elongation, Termination:

 The mRNA and aminoacylated tRNA bind to the small ribosomal unit (30S) – hamburger process
 Then large subunit (70S/50S) then binds as well  12 proteins involved in attaching small and large
ribosomal subunits
o Large subunit more difficult to determine structure
 Ribosomes 65% rRNA, 35% protein = ribozyme
o Proteins are the scaffold to hold RNA in place so RNA can carry out function
 Large ribosome active site, where peptide bond formed b/w 2 amino acids, has no proteins within
18 angstroms of that region  only RNA  evidence that RNA for catalytic function
 Erythromycin (antibiotic) blocks active site in protein exit tunnel, blocking the progression of
growing proteins
 Shine-Dalgarno sequence of mRNA in prokaryotes signals ribosome binding and guides to start
codon
o Ribosome binds the 5’ cap and tRNA and small subunit scan to find AUG with help from
Kozak consensus sequence
 A site
o Aminoacylated tRNA attaches
o Its amino end will attach to carboxyl end in P site
o A site temporarily holds peptide once peptide bond formed, then whole system slides over
 Large ribosomal subunit moves to the next codon, freeing the A site
 Small subunit moves over after
o The growing peptide from the tRNA in the P site is moved over to the newly entered tRNA
in the A site, and then the tRNA in the A site moves into the P site via peptidyl transferase
 The ATP hydrolysed for aminoacylation of the tRNA results in a high energy bond
which provides energy to generate new bond in elongation
 P site
o Growing peptide attached here to tRNA via carboxyl end
o tRNA that was holding peptide is transferred to E site
 E site

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o Exit site
o Becomes free when outgoing tRNA ejected (translocation)
 Ribosomal dissociation occurs once A site encounters a stop codon. Release factor protein binds to
the A-site and hydrolysis breaks the carboxyl bond  peptide released
o Release factor structurally similar to tRNA so it fits in the A site

 Polycistronic mRNA in prokaryotes

o One mRNA encodes multiple genes  multiple proteins translated
o 3 start codons, 3 stop codons (e.g. 1 for each gene)
 In prokaryotes, transcription and translation can be coupled in the cytoplasm at the same time

Eukaryotes:
 Ribosomes in cytoplasm and ER
 Signal sequence in mRNA determines whether a ribosome is moved from cytosol to the membrane of
the endoplasmic reticulum
o Protein fed into ER where modifications or secretions occur
o Signal peptide cut off
o E.g. Insulin produced by β cells in pancreas and has signal sequence which determined whether
it is secreted into bloodstream
o Proteins transported in vesicles to Golgi – sorting apparatus
 Cytoplasm compact with protein translation apparatus

Lecture 17: Prokaryotic Gene Expression Regulation

 Gene expression highly regulated and controlled
 All cell types have the same DNA but expressing different genes
 Regulation occurs in transcription initiation in prokaryotes
 Regulatory sequences in promoters
 E.coli needs to be versatile in gene expression to suit different environments
 Many bacterial genes are in operons
o 2-6 genes under the one promoter
o All expressed therefore regulated at once

trp Operon: anabolic

 Generating tryptophan essential for E.coli function
 Operon produces a number of polypeptides, some of which form enzymes that synthesise hormones
and eventually produce tryptophan
o Pathway not used when there is lots of trp in environment
 trp repressor made in inactive state
o trp repressor is a dimer which can bind 2 trps  elongates dimer so it binds to operon
 High [trp]  trp binds to repressor which activates it  repressor binds to operator (regulatory
sequence in E.coli promoter)
o RNA polymerase cannot get past promoter with repressor on operator  no longer making
polypeptides  no more trp produced
o Negative regulation
 Anabolic since we are making trp from gene expression

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lac Operon: catabolic

 Lac I produces active lac repressor
 In absence of lactose, repressor binds to operator of lac operon  no transcription of lacZ (β
galactosidase), lacY (permease), lacA proteins  inducible system
o Permease is a transporter protein for lactose on cell membrane
o β galactosidase can transform lactose into allolactose
 Presence of lactose/allolactose inactivates repressor (4 molecules for each subunit)  not binding to
operator  transcription occurs of lac proteins
o β galactosidase hydrolyses lactose into glucose and galactose
 Uses lactose as an energy source when genes expressed (genes break down lactose to produce
galactose and allolactose)
 Lac repressor is a tetramer but most regulatory proteins work as a dimer
o 2 dimers interact each bind to an operator site (palindromic binding sequence)
 2/3 operator sites bound
 Repressor can twist/deform DNA in order to repress

Glucose:
 Lactose takes more energy input to break down than glucose (which feeds straight into glycolysis)
 cAMP accumulates in low glucose, binds to and activates CAP (promoter upstream of RNA polymerase
binding site)  activates lac operon gene expression  positive regulation
 Low ATP = high cAMP = active CAP = expression of genes on lac operon
 Expression of lac operon never completely off, always a low level of expression

 Combinatorial control: several important regulators of a single gene, messages integrated for a single
response; sum total of all activators/repressors
 Gene activation synergistic  collectively much greater level of transcription

Lecture 18: Eukaryotic gene expression regulation

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 Regulation important in eukaryotes since many different cell types with certain necessary proteins
o Liver cell have all transcription factors required to have increased expression of albumin
o Lens cell has transcription factors for crystalline, which makes the cell transparent
 Expression of albumin in the lens would make them opaque  blindness
 Transcription factors as regulatory proteins account for 12% of genes
 Chromatin can block RNA polymerase access (eukaryotic genes more protective)
 Basal transcription is low in eukaryotes (regulation mainly of promoters), high in prokaryotes
(regulation mainly of repressors)
o Majority of regulation is positive, not negative
o Also more proteins involved in transcriptional regulation
 More transcription factors per gene, average of 6 sites

Various Regulatory Sequences:

 Can be far away upstream or in the intron of a gene
 Enhancers/enhancer elements are where activators bind
o Activators bind various amounts of genes, or may be specific - some are modified by signals
coming from outside the cell
o Activators contain zinc fingers to bind DNA enhancer elements
 Silencers are where repressors bind (not as common as activators)

Mediators:
 Mediator connects activator (with bound enhancer) and the coding region of a gene
o Histone modification/nucleosome modelling complex of mediator binds to activator
 Mediator activates TFIIB and TBP so that other proteins will bind to coding sequence, leading to
phosphorylation of the CTD of RNA Pol II  leads to transcription initiation

DNA Structure:
 Major groove more open than minor groove
 Base pairs in major groove more exposed which transcription factors recognise and bind to via DNA
binding domain
o Base pairing fit and charge more differentiable/distinct
 DNA-binding protein has more circumference within major groove to dip in and reach nucleotides
 At least two domains of protein structure; a DNA binding domain and an activation domain: modular
o Zinc finger motif has helix binding into the major groove
 Since small, they don’t recognise a big sequence, so multiple zinc fingers improve
specificity
 Zn+ co-ordinated with cysteines and/or histidines maintains structure while helix binds
in DNA pocket
 Can be many different transcription factors that have zinc fingers, since there are not
many structures that can dip into the major groove
o Helix loop helix
 Works as a dimer
 Helix binds into major groove with loop (DNA-binding domain) and then
rest of protein may lead into another domain (could interact with
mediator)
 Both helices in the dimer bind in major groove, likely to be a palindrome in the DNA
sequence

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Eukaryotic Transcription Factors:

 Large families of protein – originated from a single gene but duplication has enabled modification
o Some similarities (related sequences) between them and can work together
o Mutations causes new gene to be inactive  pseudogene
o Can end up with homodimers and heterodimers on the DNA sequence since the different
proteins have similar domains

Eukaryotic Post-Transcriptional Regulation:

 Occurs in several other steps – posttranscriptional processing, RNA stability, protein modification
 Regulation of splicing, single gene spliced to make different proteins
o Splicing repressors can bind to the pre-mRNA and alter how the spliceosome interacts with it
 Splicing repressor prevents recognition of a 3’-splice junction, so spliceosome misses
an exon and makes one big splice
o Splicing enhancers can bind to the pre-mRNA and enhance the splicing of weak splice sites
 microRNA (miRNA) discovered in 1998
o ~20 bp (20mer)
o 1/3 of genes regulated by miRNA
o Has own gene in genome, with palindromes that form loops/hairpins (2o structure)
o Once miRNA out of nucleus, dicer protein cleaves the hairpin into 2 single stranded sections
w/o hairpin
o Helicase separates the two single strands  mature miRNA loaded onto RISC protein
o Single-stranded is reverse complement of target mRNA with which it base pairs along with the
RISC protein  regulation
o If very complementary  cleaves mRNA and degraded  not translated/expressed
 Even though transcribed, mRNA not translated
o If partial complementarity, miRNA and RISC complex remain bound which inhibits translation

Post-translational Modifications:
 Formation of disulphide bridges for folding + stability
 Glycosylation (in ER) assist sin folding and stability, important for extracellular functions
 Ubiquitination – ubiquitins covalently (reversibly) add target proteins to degrade it, or can act as a
signal
 SUMOylation – addition of SUMO protein changes localisation or binding partners (function)
 Phosphorylation
o Addition of phosphate groups (charged) activates/deactivates proteins by changing
conformation
o Induces protein-protein interactions
o May change how it interacts with DNA
o Reversible modification
 Palmitoylation – covalent attachment of lipid to a protein, important if protein embeds in membrane,
used in neuronal synapses

Lecture 19: Regulation of gene expression via chromatin

 Each cell has 2m of DNA length  immensely compacted
 46 chromosomes  22 pairs of autosomes, one pair of sex chromosomes
 Chromosome: telomeres each end, centromere near middle, genes interspersed with other sequences
o One centromere for each chromosome for mitotic spindles the recognise
o Telomeres shorten with age

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 Genes stained for banding  identification

 Most compact form of chromosome during metaphase of mitosis/meiosis
o 2-8cm of DNA represented in 5-6 microns  10,000 fold compaction
o X structure = 2 identical double-stranded DNA molecules
 Most cells spend a long time in an exit phase from G1 to S phase (G0)
 Chromosomes stained in interphase show chromosomes in relaxed state, more diffuse although in
their own pockets (constrained and organised) but homologues not next to each other

DNA Supercoiling:
 DNA not a static molecule, it has a tendency to not be in a straight line
 Supercoils form to relieve tension, since DNA usually under tension (over or underwound)  regulated
by the cell
o Next step up from DNA helix
o Coil within a coil
o Most cellular DNA is underwound  makes it easier for the strands to separate e.g. for
replication
o During DNA replication, nucleosome released from histone core  DNA strongly overwound 
topoisomerase required to separate strands
 Agarose gel running DNA (same weight, same # base pairs) with different numbers of supercoils shows
ladders
o Smallest fragments with most supercoils run the fastest, go to bottom
 RNA polymerase puts tension in DNA, tension/supercoils relieved by topoisomerase
o Type I = simple, bacterial, cuts doubles stranded DNA, flips it around and re-joins
o Type II
 Multisubunit enzyme (dimer) binds a segment of a DNA molecule, with one piece to be
cut bound in the C gate
 A second segment of the same DNA molecule trapped in the N gate
 DNA in C gate is cleaved on both strands to form two 5’-phosphotyrosyl linkages with
amino acids to protect ends
 The N gate DNA segment is passed through the break
 The broken DNA in C gate is re-ligated and released error-free

Chromatin (ns) in eukaryotes:

 DNA + proteins = chromatin
 DNA protected within cell by being looped twice (~146bp) around 8 histone molecules = nucleosome
o Nucleosome = basic unit of chromatin ~ 7 fold compaction
o Nucleosome = 2 layers of 4 histones (H2A, H2B, H3, H4)
 Histones rich in arginine and lysine amino acids (basic)

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 H3 and H4 highly conserved across species

 Histones can be modified by enzymes
 H1 makes nucleosome bigger by slotting into the coil (not in core) and locking structure together
 Modifications occur on N-terminus of histone tails and body of nucleosome as well
o Histone tails are highly disordered but protect and regulate chromatin
o Some of amino acid tails interact with the tails of the neighbouring nucleosome
o Tail modification alters structure/packing  alters access of the DNA to DNA binding proteins
 Methylation, acetylation, phosphorylation of tails = part of the ‘histone code’ – recognised by proteins
which marks the DNA
o Ubiquitination occurs in core
o Protein modules bind to specific histone modifications on nucleosome
o Histone tail acetylation (by histone acetyl transferases) opens up chromatin  accessible
 Acetylation on N-terminals of tail causes positive charge to be lost  they dissociate
from DNA
o Methylation closes chromatin  inaccessible

More Packing:
 Nucleosome + H1 b/w nucleosomes form helices = 30nm fibre (~100 fold compaction)
o Packing level for DNA not being accessed by DNA binding proteins
o 30nm affected by chromatin remodelling complex which activates gene  remodelled
nucleosomes (can modify nucleosome separation), histone removal, histone replacement
o 30nm with histone-modifying enzyme  specific pattern of histone modification e.g.
methylations, phosphorylations
 X structure of chromosome has a chromosomal scaffold even when DNA not attached
 Areas of gene activity are not as tightly packed, loops have high level of gene expression
 Chromosomes move around in cell depending on how it is being expressed
o Cells have expression and repression domains

DNA Modification:
 DNA methylation has no effect on base-pairing
 De novo DNA methyltransferases (DNMT) enzyme methylates CPG dinucleotide clusters (islands) in
promoters
 Methylation represses the expression of a promoter
 Can be inherited because hemi-methylated sequences are recognised by maintenance DNA
methyltransferases, causing methylation on both sides of double stranded DNA
 Genes that make cells grow/divide often repressed by methylation rather than having a protein bound
(can still produce enzyme)

Lecture 20:
 Genome sequencing/genomics; haploid human genome ~ 3.2 x 109 base pairs
o Measuring human variation
 Human copy number variation, some people have extra or less DNA in certain regions 
people have different numbers of genes
o Medical genetics – gene data publically released, genomes compared to identify genetic
disorders e.g. Alzheimer disease
o Evolutionary past
o Human migration map
 Observing Y-chromosome or mitochondrial genomes (since they change slowly)

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o Function of a human gene/protein by comparing genomes b/w species

o Identifying novel genes
o Identifying structures in human protein
o Identifying how a gene is regulated
 First sequenced in 2001 – information was a map
 Sequencing technique improvements led to exponential increase in number of human genomes
sequenced
 Individual sequencing looks at gene markers rather than looking at the whole genome

DNA Sequencing Methods:

 Sanger sequencing – running sequences on polyacrylamide gel b/w two glass plates, tedious
 New methods (Next Gen) sequence multiple small pieces of DNA at the same time – faster
o Little pieces of sequencing (reads) matched up to reference genome

Genomes:
 Nuclear genome: 25,000 genes, 1.5% coding, lots of introns
 Mitochondrial genome
o Dense packing – little space b/w genes
o Not much regulation – expressed all the time
o Variant genetic code
 4 of the 64 codons encode a different thing compared to the nuclear genome
 Wobble in third base can be A, T, C, G  4 possible options
 Fewer tRNAs, slightly different
o Mitochondrial diseases affect energy, development, vision, cause seizures (CNS issues)
o Circular, own ribosomal RNAs, genes produce proteins for its own function
o Mitochondria requires proteins encoded by both the mitochondrial genome and nuclear
genome
 Defects can be mutations in nucleus or mitochondrial gene (maternal inheritance)

Transposons/mobile elements:
 Active piece of DNA that can move from place to place by being cut and inserted into different place
(few can do this)
 3 different types make up 45% of human genome, most located in an intron
 DNA transposons move using a DNA intermediate
 Retrotransposons move using an RNA intermediate
o LINE elements (long)
 ~6.1kb long
 Encode reverse transcriptase so can move autonomously
o SINEs (short)
 ~350bp, Alu repeats
 1.5 million copies in human genome
 Only encode a copy of themselves
 Do not encode reverse transcriptase and cannot move autonomously unless LINE
nearby with RT which can produce SINE somewhere else
 Retrovirus
o Virus injects its RNA genome and proteins into host cell
o Virus’ reverse transcriptase enzyme takes RNA and produces DNA copy, which can join with
DNA of host

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o This DNA will be transcribed producing RNA + genes  translated into reverse transcriptase
and envelope/coat  new virus particles
 Retrotransposons are an evolutionary descendent of viruses, non-infectious (can’t produce coat)

Alzheimer’s Disease:
 Before whole genome sequences and we only had maps  Pedigrees of families with early onset
Alzheimer collected
 Genomic comparison of people on pedigree using SNPs
o Single nucleotide polymorphisms (a natural change, not causing a disease)
o Observe whether SNPs relate to same location as the disease gene
o Get a region of interest, start to isolate DNA out of that and sequence it to understand where
gene is
 Identification of the chromosome and chromosomal region
 Identification of the genes in the region using the database of human genome
 Sequencing and comparison of these genes b/w people with and without early onset Alzheimer
 Identification of the presenilin-1 gene

Lecture 22: Gene Technologies

PCR:
 Amplifies one piece of DNA
 98oC denaturation  single-stranded DNA
 58oC allows annealing of DNA primers specific to the gene (~20bp long)
 72oC allows DNA Taq polymerase to add nucleotides to 3’ end of each primer (withstands high
temperatures, otherwise you have to keep adding DNA polymerase as it is degraded)
 One round doubles the amount of DNA  exponential

 Usually interested in PCR on RNA to see how much a gene is expressed

 mRNA template is annealed to synthetic oligonucleotide primer (repeated Ts binds to polyA tail of
mRNA)
 Reverse transcriptase and dNTPs yield a complementary DNA strand (mRNA-DNA hybrid)
o Rest of the process gives us double stranded stable DNA  cDNA (copy DNA)
 PCR using DNA gives us exons + introns
 PCR using cDNA gives us only the exons since mRNA only contains expressed genes
o If gene not expressed, no cDNA is produced

RT-PCR:
 Amount of cDNA produced from reverse transcriptase reflects how
much a particular gene is expressed
 When cDNA put through PCR cycle, amount of signal in exponential
phase is relative to the amount of starting cDNA
 Sample 1 has gene of highest expression since greater signal within
fewer number of cycles
 Plateau due to running out of nucleotides and primers
 RT-PCR produce can be stopped at a certain cycle and run on a gel for semi-quantitative experiment 
gel intensity indicates expression

 qRT-PCR uses fluorescent probes for a quantitative method

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 Fluorescent probe (with sequence specific for DNA of interest) has a fluorophore signal which is
quenched when the probe forms a semi-stable hairpin (reverse complement sequence)
 Probe binds preferentially to target DNA  fluorophore is separated from quenching molecule and
fluorescence signal increases
 As amount of DNA increases, more probes come out of hairpin and bind  more fluorescence
 Detector measures amount of light emitted during the reaction and indicates level of expression

Western Blot:
 SDS-PAGE gel run
o SDS = detergent unfolds proteins which separate on basis of size
o PAGE = polyacrylamide gel electrophoresis, sandwiched b/w 2 glass plates, very thin and runs
vertically
 Smaller segments run further
 Transfer proteins onto a polymer sheet, which is exposed to radiolabeled 1o antibodies which selects
gene
o Protein bands detected by specific antibodies are exposed to film
o A 2o antibody (that detects 1o antibody) produces luminescence in proportion to the amount of
protein
o Transferred to polymer sheet used where exposure to light creates image of antibodies bound
to the blot

RNA-seq database
 Whole Transcriptome Shotgun Sequencing  all of the mRNAs in a cell are observed
 RNA sample  Reverse Transcriptase  cDNA library  Next Gen sequencing of all cDNAs in
transcriptome  RNA-seq data matched to database to identify genes
 If cDNA reads are taken, then gene is expressed in the certain tissue sample
 Flat signal = no cDNA detected = gene not expressed in sample
 Lower quantity of expression = introns

Expressing Gene In Lab:

 Clone gene into a vector  expression vector so it will express in bacteria  transform into bacteria 
express the protein, purify and test it
 Plasmid vectors
o Naturally occurring circular pieces of DNA
o Antibiotic resistance gene, recognition sites for restriction enzymes, origin of replication
 Restriction endonuclease enzymes
o Recognise short palindrome sequences (~6bp) and cut in a particular pattern
o Blunt ends, sticky ends can join to compatible ends of DNA to be inserted, fragments ligated
with DNA ligase  recombinant vector
o Can also cut out DNA of phage
 Bacteria treated/transformed so they will take up, store and grow plasmid with recombinant DNA
 Introduced to antibiotics so only recombinant plasmid survives
 Bacterial promoter upstream of inserted cDNA allows expression of protein, can purify large
quantities of protein and do assays of protein activity
o Need cDNA since bacteria do not cut out introns
 Can put plasmid into human cells in culture e.g. cancer testing
 Office of Gene Technology regulator, Animal Ethics Committee

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Lecture 22: Cell Cycle Regulation

 M phase (mitosis, nuclear + cell division)
 G0 phase  exit point where cells not going through cell cycle but still carrying out their functions
o No expression of CDK and cyclin genes
o Liver cells can be induced to re-enter the cell cycle after liver damage
o Fibroblasts (skin cells for wound healing) enter and exit G0 frequently
 G1 phase  RNA and protein synthesis, no DNA synthesis
o Start in G1, restriction point. If cell passes, fully committed to S phase
 S phase  DNA synthesis, RNA and protein also synthesised
 G2 phase – RNA and protein synthesis, no DNA synthesis

Cyclin-dependent Kinases (CDKs):

 Identified by observing yeast mutants who could not move through cell cycle
 Family of protein kinases, function is to phosphorylate other proteins
 Kinase activity cyclical but stable concentration across cell cycle
 Regulates the proteins that carry out cyclical cellular functions
 Require binding to a cyclin for activity
 Animals have 8 CDKs

Cyclins:
 Undergo a cycle of protein synthesis and degradation (abundance
cycles with cell cycle)
 Essential regulators of CDK activity, also regulated themselves
 Animal have 10 cyclins
 G1/S cyclins – abundant when cell passes start
o Cyclin E + Cyclin E-CDK2
 S-cyclins – abundant for long period of time
o Cyclin A + Cyclin A-CDK 2 increases from S-phase and drops mid-mitosis-phase
 G2/M cyclins (A) – increases before mitosis then drops during mitosis
o Cyclin B increases from G2 and drops mid-mitosis-phase
o Cyclin B-CDK1 spikes during M phase
 Held inactive by phosphorylation of Tyr15 until mid-mitosis  spike

Control Of Cell Cycle:

 Phosphorylation of CDKs
o Cyclin binding to CDK moves T loop out of the active site  partially active
o Subsequent phosphorylation of Thr160 in the T-loop activates the CDK by moving Glu51 into
active site and allowing target binding  fully active
o Phosphorylation of Tyr15 near amino terminus inactivates CDK by blocking the ATP binding
site with negative charge  holds CDK in inactive state until needed
 Controlled degradation of cyclins

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o Highly controlled, precisely timed protein degradation (proteolysis)

o Occurs at G2-M transition or G1-S transition
o Dephosphorylation of Tyr15 by phosphatase makes cyclin-CDK complex active
 Complex phosphorylates targets (e.g. initiates DNA replication or mitosis)
 Complex phosphorylates phosphatase, which activates more CDK (positive feedback)
 Complex phosphorylates/activates DBRP which adds ubiquitin molecules to cyclin by
ubiquitin ligase  makes the complex a target for degradation by proteasome (negative
feedback)
o DBRP = Destruction Box Recognising Protein which binds to Destruction Box (9 amino acid
sequence near amino terminus) and causes ubiquitin to be attached
 Protein inhibitors of CDK activity (p21 or p27) bind to active site and distort it

Target Proteins of CDKs For Phosphorylation:

 Phosphorylation of nuclear lamins activates breakdown of nuclear membrane during transition into
mitosis
 Condensins are phosphorylated to compact DNA during mitosis
 Retinoblastoma protein (pRb) – checkpoint response in cell cycle
o A double-stranded break in DNA detected and information passed to ATM/ATR proteins, which
initiate DNA repair and stop the cell cycle
o p53 is activated which leads to increased p21 transcription/translation
o p21 inactivates CDK which prevents it from phosphorylating/activating its targets (e.g. pRb)
o Inactive pRb will bind to and inactivate E2F (which has downstream targets that cause cells to
undergo DNA replication)
o  CDK inactivated at a checkpoint prevents damaged DNA from replicating

o Intact DNA has no checkpoint response, no p21 produced, CDK is active and phosphorylates pRb
which now cannot bind to E2F  E2F active which activate enzymes for DNA synthesis

o Retinoblastoma = rare autosomal disease in which cancer forms in retinal cells  loss of vision
o Rb was the first tumour suppressor gene cloned and is involved in many sporadic tumours
 Rb1 negatively regulates cell proliferation, loss of function causes cancer
o Oncogenes: mutation modifies a normal gene (proto-oncogene) and now the protein signals
cell proliferation constitutively

o Knudson’s ‘2-hit’ model of tumour formation

 Hereditary: recessive mutant allele of Rb passed to daughter cells via germ line + second
somatic mutation  tumour
 Non-hereditary (less frequent): two random mutations in same somatic cell cause
retinoblastoma  tumour

Lecture 23: Signalling Pathways

 Distances:
o Contact-dependent (adjacent cells)
o Paracrine – factor released into environment which diffuses a small distance
 NB autocrine = same cell
o Synaptic – two cell bodies far apart but long axon in between
o Endocrine – gland secretes signal far away from effector tissue e.g. hormones
 Specificity: signal molecule fits binding site on its complementary receptor

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 Amplification: enzymes activate enzymes; number of affected molecules increases geometrically

 Modularity: proteins with multivalent affinities form diverse signalling complexes
 Extracellular signalling molecules usually expressed and degraded very fast  signal transient
 Same ligand causes different effects e.g. acetylcholine in heart (decreased rate and contractile force),
skeletal muscle (contraction), salivary gland (secretion)
 Molecular switches:
o Protein kinase phosphorylates ADP  ATP; system activated
o Protein phosphatase hydrolyses phosphodiester bond and dephosphorylates

G Protein-Coupled Receptors (GPCRs):

 More than 700
 7 transmembrane domain containing a protein (helical structures)
 Associated with G proteins (guanisine nucleotide-binding protein)
o Trimeric: α,γ integral membrane proteins (with lipid molecules), β
o α subunit has GDP-binding site and GTPase activity (self-hydrolyses GTP  GDP)
o Activated when GTP binds

 G protein dissociates from receptor and diffuses through membrane to activate the enzyme

β-adrenergic receptor:
 Epinephrine binds to specific receptor and sits b/w loops at tops of helices of GPCR
 G protein has bound GDP replaced by GTP  activation
 G protein diffuses through membrane and activates adenylyl cyclase, which catalyses the formation of
cAMP
 cAMP activates downstream signalling molecules e.g. PKA (cyclic AMP dependent protein kinase) which
phosphorylates cellular proteins
 GTP is hydrolysed by the protein’s intrinsic GTPase, inactive α subunit reassociated with β,γ subunits
 We have amplification: 1 epinephrine molecule  100,000 glucose molecules released into
bloodstream
o Epinephrine ligand affects 10 G-proteins which produce lots of cAMP

Receptor Tyrosine Kinases (RTKs):

 Many types, each have a single transmembrane helix + tyrosine kinase
domain in ICF, which phosphorylates tyrosine residues on specific
substrates
o Others have cysteine-rich domains, immunoglobulin-like domain (β
barrel)
 Insulin receptor has a pre-formed dimer of αβ heterodimers (4 subunits,
quaternary structure) with disulphide bridges b/w each subunit
 RTKs generally cause growth, survival  tumours occur if mutations
happen

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
o Trans triple auto-phosphorylation of carboxyl-terminal Tyr residues
o Activation loop moved dramatically in tyrosine kinase domain  space for target protein in
substrate-binding site
o Tetramer phosphorylates IRS-1 (insulin receptor substrate 1) on its Tyr residues
o SH2 domain of Grb2 binds to phosphorylated Tyr of IRS-1
 Sos bridges Grb2 and Ras, causing GDP release and GTP binding to Ras (G-protein)
o Transcription factor activated in cytoplasm, moves to the nucleus through pores  gene
expression stimulated
 Dominant negative inhibition occurs when one dimer mutated and the tetramer formed has overall no
kinase activation

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