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The CH2 chains are non-polar as C-H shares its electrons evenly.
The long chains are known as ALIPHATIC CHAINS. The longer the
chain the more hydrophobic.
CARBOHYDRATE (Hydrated Carbon) has the general formula (H-C-OH)n.
Polymer examples include cellulose, starch and glycogen. The polymer has
the general name polysaccharide (many saccharides), the monomer is a
saccharide or sugar. Sugars with 5 or 6 carbons readily cyclise forming
ring structures, the most common being glucose C6H12O6.
DNA and RNA are both Nucleic Acid. Comprises of monomers: Phosphate
group (also contains the phosphateester bond), Nitrogenous Base
(ATUGC), Sugar (ribose RNA or deoxyribose DNA). The sugar and the
phosphate group form the BACKBONE. The Base is attached to the sugar.
PROTEINS are polymers made up of the 20 amino acids. The amino acids
differ in the side chains: hydrophobic, aromatic, polar, acidic and basic.
The types of side chains and their order in the polymer determines the
nature and function of the protein as well as its shape.
THE CENTRAL DOGMA Replication by DNA polymerases, Transcription by
RNA polymerases and Translation by Ribosomes.
PEPTIDE BOND is how amino acids join together to form their chains.
They follow the general structure:
o
This bonding process is a sequetial process as more amino acids are joined
ot the growing chain. As the chain grows, we have a repeating backbone,
with an N TERMINAL and a C TERMINAL . The peptide bond formation is
extremely unfavourable as the condensation polymerisation process
releases 1 water molecule, where there is already so much water around
it. Inside the cell, water is excluded from the active site of peptide bond
formation.
SIDE CHAINS
o HYDORPHOBIC generally consist of only C-H or CH2 side chain
o AROMATIC uniquely has a carbon ring, can absorb UV light at
280nm
o POLAR non-IONIC has OH, which gives the polar property
o ACIDIC side chain contain carboxylic acid group COOH
o BASIC contains amine/amide group.
L-isomer has R group on the RIGHT, D isomer has R group on the LEFT
LEC 3
AMINO ACID CHARGE at certain pHs the amino acid will have different
charges. Eg at pH 1, it will be fully protonated, at pH14 it will be fully
deprotonated.
Charge is related to pH
o pKa is where there is 50% dissociation eg 50% of the glycine will be in
COOH form and the rest in COO form.
o There will be 2 pKas one between ve and 0 charge, then one between
0 and +ve charge.
o pI is the isoelectric point, where the charge is neutral. Calculated by
taking the average of the two pKas.
Overall charge of protein: Only side chain is considered. Using ratio of acidic
to basic residues.
o ACIDIC SIDE CHAINS: asp, glu
o BASIC SIDE CHAINS: his, arg, lys
o +acidic = -pI
o +basic = +pI
BUFFERS: Help resist changes in pH. Most buffers are weak acids or bases.
These solutions buffer best +- 1pH unit either side.
LEC 4
PROTEIN FOLDING To have a functional protein it must not only have the
correct amino acid sequence but the correct 3D formation. The main forces
which cause protein folding are the intermolecular forces:
o Intermolecular forces: Van der Waals forces, hydrophobic interactions,
Disulphide Bonds, hydrogen bonding, ionic interactions
HYDROGEN BONDING: results from a small dipole. The N terminal can act as a
H donor, and the C terminal carbonyl can act as a H acceptor. Also polar nonionic side chains can do this.
o Side chains with OH or NH all act as H donors
o Side chains with C=0 are acceptors
IONIC INTERACTIONS: Side chains with net charge +ve/-ve can interact with
each other if close enough.
VAN der WAALs INTERACTIONS: molecules that have a charge or dipole which
come close to non-polar group, induces a small dipole. This helps bring
stability to the conformation of the protein.
HYDROPHOBIC INTERACTIONS: a non-polar side chain will form a shell with
water molecules around it. This causes a loss in entropy. To minimise the loss,
the non-polar groups cluster together and removes them from exposure to
the water. This is what causes the protein to fold.
AMINO BACKBONE ROTATIONS: The peptide bond restricts the rotation. Other
bonds can freely rotate.
o
o
LEC 5
The structure of the protein is coded into the primary structure of the amino
acid sequence.
Proteins fold due to the laws of thermodynamics: 2 nd law states that
everything moves to disorder. Hence proteins fold to give it more options.
Incorrect folding can lead to diseases such as Mad Cows disease and a
number to neurological disorders.
o Chaperones are used to make sure they are folded properly and speed
up slow parts of the folding process.
o PrP (prion related protein) is an infectious agent which causes the
protein misfolding. This also causes the transmission of the disease. As
the prion infected protein is ingested, the prion can then infect other
proteins in the host.
DNA BINDING PROTEINS: can be broadly divided into 2 classes; non-base
specific, base specific
o NON-BASE SPECIFIC: these proteins dont care about the order of the
DNA bases, and act non-discriminatorily. Eg ligases, helicases
o BASE SPECIFIC: Acts on the DNA in a base sequence specific manner.
As the DNA information is inside the double helix, usually non-base
specific proteins must act on the DNA before the base-specific proteins
can.
ENZYMES: Increase the rate of reaction. They can be very specific with the
substrate they work on.
o Many bodily functions require enzymes to function correctly. Many
diseases are the result of inherited or acquired deficiencies in a
particular enzyme.
o CHARACTERISTICS: they are usually 10,000 1 million Da. Most only
require their amino acid structures to function. Others require a cofactor such as Fe, Mg, Mn or organometallic molecules called
coenzymes.
Deficiency of a cofactor/coenzyme will hence have a direct effect
on the enzyme activity.
PROSTHETIC GROUP: is a coenzyme which is covalently bonded
to a protein
HOLOENZYME: a completely catalytically active enzyme with its
cofactors
Most enzymes are named after its substrate by adding an ase
at the end.
HOW ENZYMES WORK:
o Thermodynamics vs Kinetics:
All molecules possess an intrinsic amount of internal energy due
to its molecular structure, atoms and bonds formed
Measured at standard conditions, 25 degrees, ATM 760mm Hg, 1
mole compound.
Total internal energy delta H, level of disorder energy caused by
atoms jiggle delta S
o
o
o
LEC 6
Enzymes increase the rate of reaction by lowering the activation energy delta
G
They have active sites which bind to specific substrates, in a lock +key
fashion, though not totally specific ie not a definite shape.
If free energy is positive, then the enzymes require ATP/input energy to be
catalysed
The Rate of a Reaction is measured as the #moles of product per unit time.
REACTION RATES INCREASED BY:
o Temperature,
o amount of reactant;
o catalyst
When enzyme is added, the plot of initial velocity, v, versus substrate
concentration [S] is described by a rectangular hyperbola.
o The enzyme will reach a Vmax, where the rate of reaction will pan off,
due to the enzyme active sites being saturated.
o Reaction rate is proportional to the amount of enzyme
MEASURING VMAX and Km
o Vmax is the maximum rate at which the reaction occurs
o Km is the substrate concentration at which it gives Vmax
o Km is the measure of affinity of the enzyme for the substrate
A low Km indicates a strong binding
High Km indicates a weak binding
Kcat is the number of substrate molecules converted to product per unit time
when the enzyme is at Vmax.
LEC 7
DNA
o
o
o
SUGAR
DNA contains deoxyribose, RNA contains ribose
TERMS
Base: ATGC
Nucleoside: base +sugar
Nucleotide: base + sugar + phosphate
BASES:
DNA STRUCTURE:
o Double Helix, is held together by the intermolecular weak forces
Base Stacking:
The bases are flat and aromatic (hydrophobic), hence they
can stack favourably on top of each other
Hydrogen Bonding
Hydrogen Bonding between the bases gives the DNA
double helix its specificity
Ionic/Electrostatic Interactions
Prevention of H bonding
Increased temperature
Increased pH removes the N proton from Thymine and Guanine
Formamide offers alternate H bonding
Formaldehyde reacts with the amine groups of adenine and
guanine and cytosine, prevent H bonding
Ethanol Precipitation - dehydrates DNA, removing water shell of
hydration surrounding the DNA backbone
Stability of DNA DNA must be able to store information from one generation
to the next for the next millions of years hence it must be very stable.
DNA Information Storage Properties
o Sugar Phosphate backbone very stable to chemical attack
o Absence of OH at 2 increases resistance to alkali
o N-Glycosydic bond is very stable as bases are very hydrophobic
o Base stacking excludes all water, protects the polar centre of the
double strand
o Two strands, which gives double copy and one for repair
o Hydrophilic Backbone and Hydrophobic centre makes an impenetrable
defence against any mutagens
o
LEC 8
DNA UV absorption
o An absorbance of 1 @ 260mm gives 50mg/ml DNA
o An absorbance of 1 @ 260mm gives 40mg/ml RNA
DNA negative charge
o DNA backbone has a negative charge
Detecting DNA by Fluorescence
o Can be detected by ethidium bromide
o The dye intercalates with the double stranded DNA
MELTING DNA
o By heating, the double strand becomes one strand.
o This is observed by absorbance at 260nm. The single strand will show
more absorbance, around 1.4 times more, due to more of the base
rings being exposed.
o As GC bonds are stronger, the more GC content a DNA sample has, the
higher the temperature needs to be to melt the DNA.
PROMOTING BASE PAIRS
o Use of Na+, Mg2+ shields the phosphates and thus reduce their
repulsive effects.
o EDTA buffer is used to protect the DNA from hydrolysis
DNA Interacting PROTEINS
o NUCLEASES enzymes responsible for the hydrolysis of nucleic acids
o EXONUCLEASES chew from the ends of the DNA
o ENDONUCLEASES cleave phosphodiester bonds and are sequence
specific. There are three types. We are only concerned with type II ie
REs
o RIBOZYMES RNA molecules that can cleave and reform
phosphodiester bonds
SYNTHESIS enzymes responsible for synthesising new DNA strands
o DNA polymerase I and III make new DNA from the 5 -3 diredtion
using a DNA template, primer and dNTP as substrate
o DNA Polymerases create RNA copies 5-3 direction using NTPs as
substrate. No primer
o Reverse Transcriptase Makes DNA copy from RNA template 5-3 using
dNTPs as substrate, needs primer
LEC 9
Due to the natural deamination of cytosine to uracil, Thymine is Uracil
Methylated. So that if cytosine is deaminated, then the DNA will know that it
was from Cytosine
DNA electrophoresis:
o The migration distance will depend on the number of bases in the DNA
fragment
o Seperates DNA by size
o The lighter the fragment the further it moves.
o All fragments migrate towards the positive anode
o Ethidium Bromide used to stain and view the DNA under UV light
Tm (melting temperature) is the midpoint at which double becomes single
strand
o
o
DNA REPLICATION
o The process is Semiconservative as half the original is split among the
new one.
LEC 10
DNA polymerases, not RNA poly have an editing function
o The 3-5 nuclease cleaves the newly added nucleotide if its not added
properly
All DNA polymerases need a primer
o RNA polymerases no not as they are the ones which generate the
primer for DNA poly
The need for the 3 OH for replication is exploited by drug designers
KLENOW FRAGMENT
o Comes from DNA Poly I
o By digesting DNA Poly I in protease, we get two fragments. 66kb frag is
the Klenow Frag
o It is useful as a DNA polymerase
o Needs a primer
o Very good at copying DNA
REVERSE TRANSCRIPTASE
o Produced by retroviruses
o Uses RNA template
o Produces complimentary DNA strand
o Works 5-3, needs primer
TAQ POLYMERASE
o A thermal stable DNA poly isolated from bacteria in hot spring
o Used in reaction PCR (polymerase chain reaction)
o Able to amplify sections of DNA
Full DNA replication only occurs just before cell division. The rest of the time,
DNA is being repaired.
o Must be precisely copied, once in the life time of the cell
o Only in eukaryotes.
The Elements
o Helicase unwinds DNA
Breaks the H bonds in the centre
o Single Stranded Binding Protein (SSBP) keeps the strands apart
Prevent re-annealing
o Topoisomerases untangles the supercoiled DNA
Negative Super coiling the strands have twisting energy
applied in the opposite direction as the double helix, easier to
pull apart
Positive Super Coiling the strands have twisting energy applied
in the same direction as the double helix, harder to pull apart
o Primase - lays down RNA primers
Primers are around 11-12 nucleotides in length
o DNA Pol I main copying enzyme
Copies in the 5-3 direction for the leading strand
For the lagging strand, copied in Okazaki Fragments, requiring
multiple primers
o DNA pol I fills in gaps in the lagging strand
Removes the RNA primers in the lagging strand and fills the gaps
o Ligase seals the sugar phosphate backbone
Reforms the phosphodiester bond
LEC 11 TRANSCRIPTION
The process of turning DNA into mRNA
Translation is mRNA into proteins
DNA = blueprints, mRNA = photocopy, proteins = factory
Genes determine phenotype ie skin colour, eye colour etc
Changes in genes changes phenotype and entire body development
Transcription regulates gene expression, as the amount of RNA regulates the
amount of protein produced.
Difference between prokaryotes and Eukaryotes
o Pros dont carry their DNA in the nucleus.
o Euks are all living things except bacteria
DNA is copied into RNA by RNA Pol.
o It makes a complimentary copy
o RNA has the OH at the 2 position
4 types of RNA
o mRNA carries DNA info to be translated into protein
o rRNA incorporated into the ribosome
o tRNA carries amino acids to the translation process
o small RNAs help metabolic and regulatory events
RNA Pol
o Adds one ribonucleotide at a time
o Releases PPi after each addition (each nucleotide has a tail of three
phosphate groups. PPi is the release of 2 keeping one for the backbone)
o Needs no primer
o More error prone
o 5-3 direction
Steps
o Initiation Phase RNA Pol recognises the promotor and starts
transcription
The promoter sequence is -10 TATAAT or -35 TTGACA
Promotor determines the site and direction of transcription
The closer the sequence, the stronger the binding
Genes at are copied the most will have the best match
RNA Pol then unwinds the DNA at the site called transcription
bubble
NusA and Pi leave the complex leaving RNA Pol
o Elongation Phase the RNA strand is growing
RNA Pol moves forward, copying as it goes and re-annealing
behind it
o Termination Phase: RNA Pol stops synthesis and RNA breaks off from
DNA
Near the end of the RNA copy, there is a GC rich region following
by a tail of Uracil.
The GC rich region folds up to make a hairpin.
As GC has 3 H bonds, the bonds are much stronger. The Pol
continues to read the As then drops off as the UA bonds are
weak
Some genes dont have a termination sequence. They use Rho
factor
o RHO FACTOR
Works as a hexamer
Binds to a hairpin pause site
Uses ATP for energy
Wraps the RNA chain around itself to destabilize the RNA DNA
hybrid and terminates transcription.
RNA is good as messenger as it is short lived
Good for amplification
o Thousands of proteins read from one RNA
o You can have thousands of RNA copies
DNA determines GENOTYPE
What is expressed is PHENOTYPE (proteins)
mRNA
o polynucleotide like DNA but uses ribonucleotides
o Uracil not Thymine
o Single strand
o Uses RNA Pol
o Made from DNA template
o Since it is created 5-3 the DNA template is copied 3-5
RNA Pol SUBUNITS
o Alpha, Beta, Beta Prime, omega
o Alpha associates with other proteins
o Beta catalyses the polymerisation
o Beta Prime keeps enzymes on track through DNA binding
o Omega unknown func
SIGMA SUBUNIT
o Partners with RNA Pol for transcription
o Can find the promotor sequence even though its double stranded
o Binds to promotor 10 million times the affinity of random DNA
o When coupled with RNA Pol, it is called the HOLOENZYME
TRANSCRIPTION BUBBLE
o Sigma finds promotor region and melts open the DNA
o Beta Unit catalyses the first nucleotide entry
Usually a purine G/A
A triphosphonucleotide
o As each triphosphonucleotide enters, it is connected and the PPi is
released
o 5-3 direction
o Lack of primer
o New chain is anti-parallel to DNA strand so DNA is read 3-5
ELONGATION
o The bubble moves down the DNA strand, closing and opening it as it
goes
o Around 10 nucleotides in, the Sigma unit falls off so help other RNA
coding sites
o NusA binds to RNA Pol to keep it on track of the elongation process
o RNA Pol makes a mistake every 100k 1 mill Nts
TERMINATION
o There are two ways of stopping
FACTOR INDEPENDENT
Pauses at GC rich region
As the hairpin loop forms, the RNA is pulled from the DNA
strand
FACTOR DEPENDANT
Rho Factor
Contains 6 identical subunits
Encloses the RNA
Unwinds the DNA RNA hybrid and takes out RNA Pol
LEC 12
GENE EXPRESSION
o Constitutive Passive
o Inducible/Repressible Active
Factors of Gene Expression
o Cis Factors
o Trans Factors
o Proteins
Activators
o Activating proteins often bind near the promotor
o Interacts with alpha subunit of RNA Pol to help it dock
Repressors
o Bind near promotor to block RNA Pol
Hence the balance between Activators and repressors regulates gene
expression and the rate of transcription
LIGANDS can change the activator/repressors affinity to the DNA and hence
change its activity
o Ligands can work both ways.
Ligand + activator = transcription OR
Ligand + activator = inhibitor
Similarly for repressors
o INDUCERS inactivate repressors
o CO-REPRESSORS activate repressors
POSITIVE REGULATION
o Transcription only in the presence of an activator
NEGATIVE REGULATION
o Transcription only in the absence of a repressor
OPERONS string of genes at code for enzymes
LEC 13 TRANSLATION
The conversion of RNA to Protein
Group of three bases = codon
64 different codons for the 20 amino acids
tRNA molecule is a twisty molecule which brings the anti-codon of the mRNA
molecule. It also has the corresponding Amino acid attached to it.
tRNA STRUCTURE
o Acceptor stem
Attaches to the amino acid
o T-psi-C arm
o D Arm
o Variable Arm
Anti-Codon Arm
Aminoacyl-tRNA is produced in two steps. First is the adenylation of the amino acid,
which forms aminoacyl-AMP:
amino acid + ATP aminoacyl-AMP + PPi
LEC 14
Peptide bonds form between the NH2 of the A Site amino acid and the
carbonyl carbon of the P Site amino acid
Elongation Factor EF-Tu helps move the aminoacyl-tRNA into a new site on the
ribosome
EG-G catalyses movement of the ribosome down the mRNA
Initiation Factors IF
o IF-3 helps separate the rRNA from the mRNA initially, sets the reading
frame
o IF-1 and IF-2 brings in the first tRNA.
o All IFs leave once the tRNA are in place
tRNAfmet special tRNA and amino acid used to initiate
o New proteins have N-formyl-fmet at the end
TERMINATION
o Release Factors RFs
RF-1, RF-2, RF-3
o When a stop codon enters the A Site, RFs come in.
o The peptide is hydrolysed from the P site
o All factors and rRNA leave
POLYRIBOSOMES capable of multiple translations at a time
o
o
LEC 17 CLONING
Cleaning the DNA (purifying)
SDS, Heat, Proteinase, RNase
Chloroform
Centrifuge
Aqueous Layer
Precipitate in Ethanol
Dissolve in TE Buffer
Restriction Endonucleases
Restriction enzymes in bacteria cut their DNA
and methylate them CH3 to protect them
from invading DNA
Eg EcoRI, HindIII
Many restriction enzymes cleave the DNA at
palindromic locations.
This cleaving leaves the tails
overhanging called sticky ends
PCR (polymerase chain reaction)
Amplifies DNA fragment
In cycles
Denaturation @ 95 degrees
Primer annealing @ 55 degrees
o Allows primers to hybridise their complimentary
bases
Polymerisation @ 72 degrees
o dNTPs are added to grow the chain
Each of these cycles, the DNA doubles, done 30-40 times
PCR specificity depends on the melting temperature and the
annealing temperature.
PCR
Advantages
o Quickly synthesis many copies of a target DNA
sequence
o Can use low quality DNA
Disadvantages
o Information about the target sequence must be
known
o It is sensitive to DNA contamination, as it will also
amplify impurities
Uses
o PCR cloning
o DNA sequencing
o DNA fingerprinting
LEC 18 - VECTORS
Vectors are carriers of DNA, used to be replicated and inserted into DNA
Plasmid can be used as a vector
o Effective as it only has one recognition sequence
o Has multiple cloning sites
o REs are used to cleave out a space for the concerned DNA fragment we
want to clone
Primers add the respective sticky ends onto the DNA frags so
that they can be inserted
Compatible ends are joined with DNA ligase
Ligase only joins compatible ends
o Needs an OriV, origin of replication. V stands for Vegetative which
allows it to be copied to new cells
o Not all new plasmid cells will have the recombinant DNA in it
Ampicillin is used to kill off the plasmid cells which dont have
the DNA fragment
The ones which do have the DNA wouldve had a resistant gene
inserted with the DNA ie ampR
o The filtered cells need to be further filtered, as some will now contain a
wrong DNA fragment.
Electrophoresis is used to find the correct colony of correct DNA
frag
o Then it is PCR amplified
LEC 19
The insulin gene is cloned using plasmids for use
cDNA is DNA which is created from mRNA via reverse-transcription
MAKING CDNA
o Isolation of mRNA
o Application of reverse transcriptase; RNA dependant DNA Pol
o Amplification vie PCR and Taq Pol
o Ligate into vector, transform into bacteria to colonise
TERMINOLOGY
o Locus location of a gene or DNA sequence
LEC 20
TERMS
o Substrate compound acted on in a reactions
o Product compound produced in the reaction
o Metabolism all biochemical reactions in the cell
In the human body, Genes code for enzymes used in the body. These
enzymes work together in a chain to produce a product in the end line.
Mutations cause a gene to reshuffle, hence an enzyme doesnt work anymore.
This breaks the chain and the end product cannot be created, leading to
disease.
Eg In dietary proteins, Phenylalanine is present. Phenylketonuria is the
metabolic disorder where the body cannot break down enough of the
phenylalanine and the concentration reaches toxic levels. Aka deficiency in
phenylalanine hydroxylase.
GALACTOSEMIA
o Lack of the enzyme: galactose 1-phosphate uridyl transferase
o Inability to break down galactose (subunit of lactose sugar, found in
milk)
o Causes dehydration, loss of appetite, cataracts, retardation
o Must be treated within first few days of birth
It is AUTOSOMAL RECESSIVE
Meaning you require two of the recessive genes to fully affect.
If a recessive is paired with a dominant then the enzyme activity
will be lower but still functional
THALASSEMIA
o Haemoglobin transports oxygen, comprised of 4 globin protein
molecules, two alpha, two beta, each with an iron heme group
o Imbalance in the relative amounts of the alpha beta globins produced
Alpha thalassemia
Beta thalassemia
o Types of Alpha thalassemia
Normal 2 copies of alpha globin
Alpha-Thal-2 deletion of one copy
Alpha-Thal-1 deletion of both copies
o Beta Thalassemia
Caused by pseudogenes, which are non-functional and prevents
expression
GENE ALLELES
o Can change over the course of a species lifetime
o Eg aging factors, changes hair/fur pigments
o Alleles at one locus can affect the alleles at another locus/loci
PHENOTYPES
o DISCONTINUOUS distinct variation eg short/tall peas
o CONTINUOUS overlapping egheight of humans
GENOTYPE + ENVIRONMENT = PHENOTYPE
Mutations can block biochemical pathways (PKU, galactosemia)
Genes can have multiple alleles (galactosemia)
Genes expression can change during development (hemoglobin)
The alleles at one locus can affect the alleles at another locus
The disCncCon between conCnuous and disconCnuous traits
Polygenic inheritance
Complex traits
Threshold traits
Effect of environment
o