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BIO130H1 Lecture Notes - Dna Replication

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Jane Mitchell

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Lecture 3: Introduction to Nucleic Acids & Proteins
1. Molecular interactions
2. Properties of nucleic acids
3. Intro to proteins
4. Amino acid structure
5. Protein structure
Molecular interactions
oThe types of interactions that occur between macro-molecules (DNA, RNA,
protein) are transitory(dynamic). They are different from types of interactions
that occur between the various monomers that make up nucleic acids proteins
oPhosphodiester bonds: the linkages between individual nucleic acids in DNA
chain - covalent linkages, very hard to break apart and provide stability
oElectrostatic interactions: ionic bonds, found in cellular environments, useful
but only in specific interactions inside the cell
oHydrogen bonds: formed between proton donors and acceptors, a lot of
interactions that occur between macro-molecules are engendered by these
hydrogen bonds interactions
oVan der Waals attractions: pulling macro-molecules together
oHydrophobic forces: combination of hydrophilic coming in close proximity with
hydrophobic cells, the act of living in the aqueous environment of a cell tends
to push hydrophobic into entities closer together
oThese are the types of interactions that are involved in stabilizing macro-
molecules and allowing for them to interact in a dynamic fashion
oWe talk about each one of these linear pieces of information having polarity.
The polarity resides within the chemical groups associated with either ends
oThe 5’ end represents the free phosphate group which is found on the first
nucleotide of a particular chain that is being formed
oThe 3’ end refers to that free hydroxyl group
o5’ to 3’ orientation occurs in DNA, RNA, protein
oEach one of these macro-molecules have specific orientation: linear
information, specific polarity(orientation)
Nucleic acid chains
oDNA is synthesized from deoxyribonucleoside triphosphates, otherwise known
as dNTP’s
oRNA is synthesized from ribonucleoside triphosphates, or NTP’s
oNucleotides are linked by phosphodiester bonds
oThe chemistry involved in DNA replication is a function of 5’ to 3’ orientation. It
also describes the directionality of a lot of processes involved in replication and
transcription. The biochemistry ONLY works in this specific orientation and the
enzymes involved in the biochemistry recognize this orientation
Base pairing
oHydrogen bonding occurs between bases on individual chains of DNAs and
RNAs: A-T, G-C
oThe way the bases are oriented on these DNA strands allow for 2 hydrogen
bonds to form between A and T, 3 to form between G and C
oBy having pyrimidines pairing with purines, you maintain same sort of
diameter of the double helix and in shape
Three forces that keep DNA together…
oHydrogen bonding occurs in a linear direction between the strands. They are in
their strongest configuration when they are forming in a straight line
oVan der Waals attractions: flickering dipoles between these macro-molecules
allows for helix to get squeezed together in a tighter configuration
oHydrophobic interactions: tries to avoid hydrophilic environment
DNA structure
oThe helix is the most energetically favored configuration for this double
stranded molecule with its hydrophobic and hydrophilic entities
oThe hydrophilic entities are the sugar-phosphate backbones on the outside.
They are highly charged and like to be presented to the aqueous phase
oThe hydrophobic entities likes to avoid aqueous phase
oThe major groove has a real functional significance provides access by
proteins (transcription factors, DNA modifying enzymes) to the array of bases
which are inside of the helix and are able to make hydrogen bonds with
specific bases sequences
oThe minor groove is quite narrow and doesn’t provide sufficient access for
proteins to enter into the interior of that helix
oThe strands in a double helix are ranged in antiparallel configuration (5’ to 3’
in one orientation and 3’ to 5’ in other orientation)
oThose 5’ and 3’ components are a function of the terminal groups on those
o5’ = phosphate group
o3’ = free hydroxyl group
oThe sequence of the two strands are complementary
oFidelity(the sense of going from one generation to the next) is extremely
important so that you can fix the mutation by looking at another copy
oYou can renature the DNA such that it will find its complementary copy
oYou can denature DNA(separate them out into 2 separate strands) simply by
changing the temperature as you increase the temperature, the hydrogen
bond breaks down, the DNA will peel apart from one another and form 2
separate single-stranded molecules
oThese are important for DNA replication and RNA synthesis
Unzipping the helix
oHeating denatures double stranded DNA by disrupting the H-bonds
oThe temperature at which DNA denatures is called the melting temperature
and it varies between species
oDenaturation of DNA is a reversible process heat it and separate it into 2
strands, cool it and the 2 strands will re-associate
Application of DNA denaturation/renaturation capacity
oA mechanism where you can take very small amounts of DNA and amplify it
oWe heat a piece of double stranded DNA (genome) and separate into 2
separate strands and design small aligomer primers that are complementary
to each other strand
oThose primers will add nucleotides and make complementary copy of those
separated strands, so there are now 2 double stranded DNA strands
oThen we do another round with another temperature increase
oThere are a lot of primer in the solution and that will go for more DNA
oYou will do that for approximately until 18 double strands are formed or
however long you want to do, and you will end up with a DNA sample that is
highly rich in that specific stretch of DNA between the primers
Typical PCR thermocycler
oThe DNA are placed in the microtubes inside the thermostated vessels
oThere are cooling/heating plate underneath that allows you to carefully
regulate the temperature in a programmable fashion
Introduction to protein structure
oThey are aligomers of primary units
oPrimary sequences: specific arrays of amino acids that make up that protein
oSecondary organization: capacity of single polypeptide chain to form 2 or 3
standard structures(helix, sheet) and involves local folding
oTertiary(long-range folding): overall organization of single polypeptide chain
how it folds up
oQuaternary: interactions of multiple polypeptide chains
oSupramolecular: many copies of particular polypeptide
Protein structure
oThe amino acid side-chain, or R group is variable and determines the type of
amino acid, the functional aspect of what that amino acid does
oNucleic acids come in 4 different flavors: basic(+), acidic(-), uncharged polar
(have capacity to form a hydrogen bond), and non-polar (hydrophobic) – these
charges are associated with R groups
oAmino acids come in 20 different flavors
Examples of basic and acidic AAs
oHistidine is positively charged - basic
oAspartic acid is negatively charged – acidic
oThe charges determine whether the AAs will bond with cations, anions, etc.
Examples of polar and non-polar AA’s
oThreonine is polar
oAnd phenylalanine is non-polar
Cysteine and disulphide bonds
oCysteine has the capability of responding to the redox(oxidation or reduced)
environment cells and it does this through oxidation or reduction of the
sulpherhydro group under reduced condition it is SH group and under
oxidized condition it can interact with another cysteine residue and form a
disulphide bridge (strong covalent bond)
oAll this responds to the redox environment of the cell
Structure of the genetic code
oA lot of amino acids are grouped in specific sector such that they form a tight
associations where hydrophobic, hydrophilic, polar, non-polar, charged, un-
charged amino acids seem to have similar types of codon structures
Mutational steps between codons
oNumber of mutations between codons for different AA’s are the minimum
number of mutational steps between amino acids
oTo get from a codon for proline to cysteine, you need 2 mutations and
maximum number needed is 3
oGroups of AA”s with similar properties tend to be clustered in the codon table
oCodons of AA’s with similar properties tend to have fewer mutational steps
between them
oOne random mutation in a codon is less likely to result in a dramatic change in
AA properties than 2 random mutations
Synthesis of proteins
oInvolves a condensation mutation between AA we call it condensation
reaction because there’s a production of water during the formation of peptide
oThe peptide bonds are formed between terminal group of carboxide group of 1
AA and terminal aiming group of adjacent AA
oThe result is linear aligomer that has polarity one end is going to be aiming
group always and the other end is going to be carboxyl
The order of AA’s is important
oWe always write polypeptide sequences with the N term to the left and C term
to the right
oOrientation counts as does the sequence
The alpha helix
oThe hydrogen bonds that stabilize alpha helix occur between the amides and
carboxyl group within the interior of the helix
oThe R groups are off to the side and are not part of the hydrogen bonding