DNA and RNA: Composition and Structure

Report
TOPIC 2: BIOMOLECULE 1
DNA & PROTEIN
1
Genetic information
usually
maybe
Deoxyribonucleic acid
Ribonucleic acid
Transcribed to
Held together by
Can
become
Made of
Made of
Double helix
Stabilized by
has
Single
stranded
e.g
Denaturation/
Replication
Exist
in
Hydrogen
bonds
Is three main
Linked nucleotides
has
has
Anti parallel
strands
sugar
bases
Phosphate ester link
e.g
bases
Several forms
e.g
Structural
hierarchy
B, A, Z
Adenosine
Cytosine
Guanine
Thymine
Uracil
rRNA
tRNA
mRNA
Participate in
Contains code
for
translation
Is synthesis of
Protein
Primary
is
sequence
Secondary
is
3D confirmation
Tertiary
e.g
e.g
Supercoiling
Histones
e.g
Condense DNA in
chromatin
2
DNA overview
• Hallmark of life – the ability to produce
• The unique information for each individual
must be preserved and passed to progeny
• All life on earth uses nucleic acids for
storage genetic information
• Except for viruses; all life use deoxyribonucleic
acid (DNA) to store information
3
Central dogma of Molecular Biology
•Sequential genetic information
transferred from DNA residue to
synthesis protein
•DNA play essential role in heredity by
serving as template for its replication.
•DNA cannot flow directly to synthesis a
protein
•Genetic information from DNA is
transferred to RNA through
transcription
•The sequence of RNA is translated into
a protein sequence
4
Structure and components of the
nucleotides
• Nucleic acid consist of nucleotide monomer
5
Nucleic acids consist of repeating nucleotide that have
phosphate ester, a pentose sugar, and a heterocyclic base.
Phosphate
Group
O
O=P-O
O
5
CH2
O
N
C1
C4
Sugar
(deoxyribose)
C3
C2
Nitrogenous base
(A, G, C, or T)
6
Nucleoside
• Base bound to pentose
sugar
• Pentose sugar attached
to ribose –
ribonucleoside
• Pentose sugar attached
to deoxyribosedeoxyribonucleosides
NH2
N
O
O
O
-
P
O
O
-
CH2
N
O
OH
d e o x y c ty id in e m o n o p h o s p h a te (d C M P )
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Types of nucleic acid
• DNA – Deoxyribonucleic acid
• RNA – ribonucleic acid
H OC H 2
O
OH
rib o s e
OH
OH
H OC H 2
OH
O
OH
(n o O )
d e o x y rib o s e
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Structure and component of the
nucleotides
• Nucleotide also called as nucleic acid base
• Base- refer to the nitrogen aromatic
compound
• Nucleic acid base type: pyrimidine and purine
• Pyrimidine – single ring
• Purine – double ring
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Pyrimidine and Purine
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Nitrogen-Containing Bases
O
NH2
H
N
N
N
N
N
O
N
H
H
a d e n in e (A )
th ym in e (T )
O
H
NH2
NH2
N
N
N
CH3
N
H
g u a n in e (G )
CH3
N
O
O
N
H
c yto s in e (C )
Uracil generally only in RNA
Thymine generally only in DNA
H
CH3
N
O
N
H
u ra c il (U )
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Nucleosides in DNA
Base
Sugar
Adenine (A) Deoxyribose
Guanine (G) Deoxyribose
Cytosine (C) Deoxyribose
Thymine (T) Deoxyribose
Nucleoside
Adenosine
Guanosine
Cytidine
Thymidine
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Nucleosides in RNA
Base
Adenine (A)
Guanine (G)
Cytosine (C)
Uracil (U)
Sugar
ribose
ribose
ribose
ribose
Nucleoside
Adenosine
Guanosine
Cytidine
Uridine
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Formation of Nucleic Acid Structure
• Polymerization nucleotides
form nucleic acids
• The phospodiester links the
5’OH of one residue and
3’OH of the next
• One end must terminate at
in 5’OH, the other
terminates at 3’OH.
NH2
Β-glycosidic bond
N
CMP
O
O
O
-
P
O
O
CH2
-
O
NH2
3
OH
N
N
P
O
N
N
O
O
3’-5’ Phosphodiester
bonds
N
O
-
5
CH2
O
AM P
OH
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Nucleic acid structure
Single letter represent
individual base
Sequence of bases are unique
and make each of us different!
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Double helix structure of DNA
•
•
•
•
•
•
•
Determination of double helix structure was
based on the X-ray diffraction patterns
Amount of T equal to A
Amount of G equal to C
Consist of 2 polynucleotide chain (we call it
DNA Strand) that wrapped to each other to
form helix
Chain run in antiparallel directions:
 5’ to 3’ – sense strands
 3’ to 5’ – antisense strands
Sugar phosphate backbone – outer part
Bases pair is complementary:
 A—T (2 H bond)
 G – C (3 H bond)
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Unwinding the helix
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Complementary base pairing
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DNA Sequence
Length of DNA sequence depends on organism:
Bacteria e.g. Salmonella ~ 4Mb (4 million)
Human ~ 3.4 Gb (billion)
•
•
•
kb (= kbp) = kilo base pairs = 1,000 bp
Mb = mega base pairs = 1,000,000 bp
Gb = giga base pairs = 1,000,000,000 bp.
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DNA sequence
• DNA sequence is obtained through Sequencing method
• Sequence can be uploaded in NCBI database
• Sequence of interest can also be found in the website
We will find time to review this webpage later…
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Denaturation of DNA
• A process by which double stranded
DNA unwinds and separates into
single stranded strands through the
breaking of hydrogen bonding
between the bases
• Can be achieved through heating the
DNA in solution
• Complete denaturation- ~94°C
• Temperature needed depends on the
base content of DNA; High G-C
content will need a higher
temperature. And why?
21
Denaturation of DNA
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Renaturation of DNA
Reformation of complementary strands that were separated
by heat by slow cooling process
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We will discuss on DNA compaction next week!
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PROTEIN 1:
COMPOSITION AND
STRUCTURE
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Functional role of proteins in
mammalian organism
• Catalysis in chemical transformations-enzymes
• Transport –
 Hemoglobin and myoglobin transport O2 in blood
and muscle
 Transferrin transport iron in blood
• Metabolic control- enzymes involve in the process
• Contraction – myosin and actin function in muscle
contraction
• Matrix for bone and connective tissue – collagen and
elastin form the matrix of bone and ligament
• Α-keratin- in hair and other epidermal tissue
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AMINO ACID COMPOSITION OF
PROTEINS
• All different type of proteins are synthesized
as polymers of only 20 amino acids
R Group- uniquely define each
of amino acid
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R Groups of Amino Acids
Is used to classify amino acids:
• Polar or non polar
• Acidic or basic
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Polar uncharge
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Non polar hydrophobic
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Acidic
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Basic
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Abbreviations
34
Amino acids can act as both acids and
bases
CO2H – Can be deprotonated to become
negative carboxylates (COO-) – cause
acidity
NH2- – Can be protonated to become
positive α- ammonium groups (+NH3) –
cause basic properties
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Amino acid as zwitterion
Basic group
Acidic group
H transfer
Zwitterion – a condition when amino acids are without charged groups on
their side chain – no net charge; in solution- neutral
36
Adding alkali to amino acid solution
+ [OH+]
Donate > [H+] to bind with
+[OH+]
NH3+ become
NH2 ( only –ve charge in
COO- left)
Now this aa in
negative charge!
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Adding acid to acid amino solution
+ [H+]
Now this aa in
positive charge!
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Amino acid charge
General condition
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Amino acids are polymerized into
peptides and proteins
• Isoelectric pH- the pH at which a molecule has
no net charge – also called as isoelectric point
(PI value)
• The PI- allow protein to be separated using
electrophoresis, isoelectric focusing and ion
exchange chromatography
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• TASK 1: Explain the function of plasma protein
in diagnosis of animal disease
*Must include charge interaction and
electrophoresis idea
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Two shape of proteins:
• Fibrous protein
• Globular protein
 Fibrous protein
 Provide mechanical support
 Often assembled into large cables or threads
e.g: α-keratin – major components of hair and nails
collagen – major components of tendons, skin,
bones and teeth
 Involved in structure :tendon, ligaments, blood clots –
collagen and keratin
 Contractile protein in movement: muscle, microtubule
(cytoskleton, mitotic spindle, cillia, flagella)
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Globular protein
• Usually water soluble, compact roughly spherical
• Hydrophobic interior, hydrophilic surface
• Globular protein include enzyme carrier and
regulatory protein
• Most protein which move around (e.g albumin,
casein in milk)
• Proteins with binding site:
 Enzymes, haemoglobin, immunoglobulin,
membrane receptor sites
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The peptide bond
• Peptide- short polymers of amino acid monomers
linked by peptide bonds
• Polypeptide chain – longer peptide chain
44
Hierarchy structure of protein
Primary structure (A.A sequence)
Secondary structure (α-helix and β-pleated sheet)
Tertiary structure (3-D structure formed by assembly of
secondary structure)
Quaternary structure (structure formed by more than
one polypeptide)
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Primary structure of proteins
Sequence of amino acid in polypeptide chain
Is held together by peptide bonds
Two ends – N terminus and C terminus
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Secondary structure
• Local 3-D folding of the
polypeptide chain in the
protein
• Arrangement in space
of the atoms in the
peptide backbone
• Two type: α-helix and βpleated sheet
α-helix
β-pleated sheet
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Secondary structure
• Forces involve:
Strong – covalent bond
Weak – hydrogen bond, electrostatic
interactions, hydrophobic effect
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Tertiary structure
• 3D arrangement of all atoms in the proteins, including
those in side chains and in prosthetic group
• Describes the folding and other contortions of a
polypeptide chain that result from the molecular
interactions among the R groups of the different amino
acids
• The folding is sometimes held together by strong
covalent bonds (cystein-cystein disulphide bridge)
• 3-D structure is determined through X-tray
crystallography
• Now can be predicted using bioinformatic technique
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Forces involved in tertiary structure
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Quaternary structure
• Arrangement of polypeptide chains in a multi
chain protein
• The chain is called subunit
• Subunit must be in non covalent association,
maybe connected by disulfide bonds
• Not all protein have this structure
• E.g.
chymotrypsin contains 3 polypeptide joined
together by interchain disulfide bonds
Hemoglobin – bohr effect
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DENATURATION AND REFOLDING
• Non covalent interactions that
maintain 3-D structure of protein
are weak – can be disrupted
easily
• Unfolding a protein (i.e.
disruption of tertiary structure)denaturation
• Denaturation and reduction of
disulfide bonds- happen when
complete distruction of tertiary
structure is desired
• Disruption process can be
recovered - refolding
52
Denaturation
Can be achieved in two ways:
1. Heat – increase temperature trigger vibration
in molecules – when energy become great
enough can disrupt the tertiary structure
2. At extreme high or low pH- some charges will
be missing- so electrostatic interactions that
normally stabilize the native and active form
of protein are drastically reduced
53
Denaturation
3. Detergents –

binding of detergents (e.g. sodium
dodecyl sulfate)- can disrupt
hydrophobic interaction

charged detergent – disrupt
electrostatic interactions

urea / guanidine hydrochloride –
form hydrogen bonds with protein
that stronger than internal hydrogen
bond

B- mercaptoethanol – reduce
disulfide bridge to 2 sulfuhydryl
groups
#what do they use to straighten your
curly hair?
54
Relationship between protein
structure and its function
• Protein structure determines protein function
• Denaturation or inhibition may change protein
structure - will change its function
• Coenzyme and co factor may enhance the
protein’s structure
55
Protein folding
• A process in which a
polypeptide folds into a
specific, stable, functional 3-D
structure
• In order to carry out their
function (e.g. enzymes or
antibodies), protein must take
on a particular shape, also
known as ‘fold’ – from 1° to 3°
• Thus protein are amazing
machine! Before they do their
work, they assemble
themselves! This selfassembly is called ‘folding’
56
The importance of correct folding
• Primary structure carry all the information needed to
produce correct tertiary structure – but the process it
self sometimes not straight fwd and trickier
• Protein dense environment cell – protein may fold
incorrectly as they produced
• Or they may begin to associate with other protein
before they complete their own folding
• In euk – proteins may need to remain unfolded long
enough to be transported across the membrane
• Correctly folded – usually soluble in aqueous cell
environment or attached in membrane
57
The importance of correct folding
• Folding incorrectly –
may interact with other
proteins and form
aggregates (Accumulate
and clump together )
• Fail to do so –
ineffective use of
protein or producing
toxic protein! – lead to
protein folding disease
58
The importance of correct folding
• Occur because
hydrophobic regions
that should be buried in
side the protein remain
exposed and interact
with other hydrophobic
regions of other
molecules
Hydrophobic chain –
red color – interior
Hydrophilic chain –
green color –exterior
59
Protein folding chaperone
• Chaperone – special protein that help in
correct and timely folding protein
• Prevents protein form associating with
another protein or prevent it from associating
with itself in inappropriate way
• E.g. of chaperone – hsp 70 (70 kilodalton heat
shock protein)
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Chaperon helps in
formation of hemoglobin
• Hb – consist of α globin and β globin
chain
• Produced by α and β globin gene
• There are 2 α globin gene for one β
globin gene – thus there is excess of α
globin chain – aggregate among
themselves damaged RBC – thallesemia
– useless form of hemoglobin
• Therefore, α globin chain need to kept
from aggregating – so they are enough
in to complex with β globin chain
• With help from chaperone – α
hemoglobin stabilizing protein – prevent
α globin chain from causing damaged to
RBC and help to deliver them to β globin
chain
61
TASK 2 – DISCUSS THE EXAMPLE OF PROTEIN
FOLDING DISEASE BY STATING THE
MECHANISM
62

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