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Nucleic Acids
Nucleic Acids Are Essential For
Information Transfer in Cells
• Information encoded in a DNA
molecule is transcribed via synthesis
of an RNA molecule
• The sequence of the RNA molecule
is "read" and is translated into the
sequence of amino acids in a
protein.
Central Dogma of Biology
Nucleic Acids
• First discovered in 1869 by Miescher.
• Found as a precipitate that formed when
extracts from nuclei were treated with
acid.
• Compound contained C, N, O, and high
amount of P.
• Was an acid compound found in nuclei
therefore named nucleic acid
Nucleic Acids
• 1944 Oswald, Avery, MacLeod and
McCarty demonstrated that DNA is
the molecule that carrier genetic
information.
• 1953 Watson and Crick proposed
the double helix model for the
structure of DNA
Nucleic Acids
• Nucleic acids are long polymers of
nucleotides.
• Nucleotides contain a 5 carbon sugar, a
weakly basic nitrogenous compound
(base), one or more phosphate groups.
• Nucleosides are similar to nucleotides
but have no phosphate groups.
Pentoses of Nucleotides
• D-ribose (in RNA)
• 2-deoxy-D-ribose (in
DNA)
• The difference - 2'OH vs 2'-H
• This difference affects
secondary structure
and stability
Nitrogenous Bases
Bases are attached by b-Nglycosidic linkages to 1 carbon of
pentose sugar – (Nucleoside)
Nucleosides
• Base is linked via a b-Nglycosidic bond
• The carbon of the glycosidic
bond is anomeric
• Named by adding -idine to
the root name of a pyrimidine
or -osine to the root name of
a purine
• Conformation can be syn or
anti
• Sugars make nucleosides more
water-soluble than free
bases
Anti- conformation
predominates in nucleic acid
polymers
Nucleotides
• Phosphate ester of nucleosides
The plane of the base is oriented
perpendicular to the plane of the
pentose group
Other Functions of
Nucleotides
• Nucleoside 5'-triphosphates are
carriers of energy
• Bases serve as recognition units
• Cyclic nucleotides are signal molecules
and regulators of cellular metabolism
and reproduction
• ATP is central to energy metabolism
• GTP drives protein synthesis
• CTP drives lipid synthesis
• UTP drives carbohydrate metabolism
• Nucleotide monomers are joined by 3’-5’
phosphodiester linkages to form nucleic acid
(polynucleotide) polymers
Nucleic Acids
• Nucleic acid backbone takes on
extended conformation.
• Nucleotide residues are all oriented
in the same direction (5’ to 3’)
giving the polymer directionality.
• The sequence of DNA molecules is
always read in the 5’ to 3’ direction
Bases from two adjacent DNA
strands can hydrogen bond
•Guanine pairs with
cytosine
•Adenine pairs with
thymine
Base pairing evident in
DNA compositions
H-bonding of adjacent antiparallel
DNA strands form double helix
structure
Properties of DNA Double Helix
• Distance between the 2 sugar-phosphate backbones
is always the same, give DNA molecule a regular
shape.
• Plane of bases are oriented perpendicular to
backbone
• Hydrophillic sugar phosphate backbone winds around
outside of helix
• Noncovalent interactions between upper and lower
surfaces of base-pairs (stacking) forms a closely
packed hydrophobic interior.
• Hydrophobic environment makes H-bonding between
bases stronger (no competition with water)
• Cause the sugar-phosphate backbone to twist.
View down the Double Helix
Hydrophobic
Interior with base
pair stacking
Sugar-phosphate
backbone
Structure of
DNA Double
Helix
• Right handed helix
• Rise = 0.33
nm/nucleotide
• Pitch = 3.4 nm /
turn
• 10.4 nucleotides
per turn
• Two groves – major
and minor
• Within groves,
functional groups on
the edge of base
pairs exposed to
exterior
• involved in
interaction with
proteins.
Factors stabilizing DNA
double Helix
• Hydrophobic interactions – burying
hydrophobic purine and pyrimidine rings
in interior
• Stacking interactions – van der Waals
interactions between stacked bases.
• Hydrogen Bonding – H-bonding between
bases
• Charge-Charge Interactions –
Electrostatic repulsions of negatively
charged phosphate groups are minimized
by interaction with cations (e.g. Mg2+)
DNA
• 1o Structure - Linear array of
nucleotides
• 2o Structure – double helix
• 3o Structure - Super-coiling, stemloop formation
• 4o Structure – Packaging into
chromatin
Determination of the DNA 1o
Structure (DNA Sequencing)
• Can determine the sequence of DNA
base pairs in any DNA molecule
• Chain-termination method developed
by Sanger
• Involves in vitro replication of
target DNA
• Technology led to the sequencing of
the human genome
DNA Replication
• DNA is a double-helical molecule
• Each strand of the helix must be copied in
complementary fashion by DNA polymerase
• Each strand is a template for copying
• DNA polymerase requires template and
primer
• Primer: an oligonucleotide that pairs with
the end of the template molecule to form
dsDNA
• DNA polymerases add nucleotides in 5'-3'
direction
Chain Termination Method
• Based on DNA polymerase reaction
• 4 separate rxns
• Each reaction mixture contains dATP, dGTP,
dCTP and dTTP
• Each reaction also contains a small amount of
one dideoxynucleotide (ddATP, ddGTP, ddCTP
and ddTTP).
• Each of the 4 dideoxynucleotides are labeled
with a different fluorescent dye.
• Dideoxynucleotides missing 3’-OH group. Once
incorporated into the DNA chain, chain
elongation stops)
Chain Termination Method
• Most of the time, the polymerase uses
normal nucleotides and DNA molecules
grow normally
• Occasionally, the polymerase uses a
dideoxynucleotide, which adds to the
chain and then prevents further growth
in that molecule
• Random insertion of dd-nucleotides
leaves (optimally) at least a few chains
terminated at every occurrence of a
given nucleotide
O
O
N
NH
N
NH
N
NH2
N
N
HO
NH2
O
H
HO
H
N
H
O
H
H
N
H
P
NH2
O
N
H
O
NH2
N
H
O
N
O-
N
H
H
N
O
P
N
O-
N
O
O
H
O
H
H
O
O
H
H
H
N
O
NH
O
O
PH
N
O-
N
H
P
N
O-
NH2
O
O
O
O
O
P
O
H
O-
OH
H
H
H
OH
H
H
H
H
O
H
NH
H
OH
OH
P
H
N
H
OH
O
O
H
N
NH2
O
N
NH
N
HO
NH2
N
NH2
O
H
H
N
H
O
O
N
H
H
P
N
O-
NO CHAIN
ELONGATION
N
O
O
H
H
H
O
H
H
N
OH
O
P
O
P
PH
N
O-
O
O
O
O
O-
H
H
OH
H
H
H
O
NH
OH
N
NH2
Chain Termination Method
• Run each reaction mixture on electrophoresis gel
• Short fragments go to bottom, long fragments
on top
• Read the "sequence" from bottom of gel to top
• Convert this "sequence" to the complementary
sequence
• Now read from the other end and you have the
sequence you wanted - read 5' to 3'
DNA Secondary structure
• DNA is double stranded with
antiparallel strands
• Right hand double helix
• Three different helical forms (A, B
and Z DNA.
Comparison of A, B, Z DNA
• A: right-handed, short and broad, 2.3 A,
11 bp per turn
• B: right-handed, longer, thinner, 3.32 A,
10 bp per turn
• Z: left-handed, longest, thinnest, 3.8 A,
12 bp per turn
A-DNA
B-DNA
Z-DNA
Z-DNA
• Found in G:Crich regions of
DNA
• G goes to syn
conformation
• C stays anti
but whole C
nucleoside
(base and
sugar) flips
180 degrees
DNA sequence Determines Melting Point
• Double Strand DNA can be
denatured by heat (get strand
separation)
• Can determine degree of
denturation by measuring
absorbance at 260 nm.
• Conjugated double bonds in
bases absorb light at 260 nm.
• Base stacking causes less
absorbance.
• Increased single strandedness
causes increase in absorbance
DNA sequence Determines Melting Point
• Melting
temperature
related to G:C and
A:T content.
• 3 H-bonds of G:C
pair require higher
temperatures to
denture than 2 Hbonds of A:T pair.
DNA
o
3
Structure
• Super coiling
• Cruciform structures
Supercoils
• In duplex DNA, ten bp per turn of helix (relaxed
form)
• DNA helix can be over-wound.
• Over winding of DNA helix can be compensated by
supercoiling.
• Supercoiling prevalent in circular DNA molecules
and within local regions of long linear DNA strands
• Enzymes called topoisomerases or gyrases can
introduce or remove supercoils
• In vivo most DNA is negatively supercoiled.
• Therefore, it is easy to unwind short regions of
the molecule to allow access for enzymes
Each super coil compensates for one + or – turn of
the double helix
•Cruciforms occur in
palindromic regions of DNA
•Can form intrachain base
pairing
•Negative supercoiling may
promote cruciforms
DNA
o
4
Structure
• In chromosomes, DNA is tightly
associated with proteins
Chromosome Structure
• Human DNA’s total length is ~2 meters!
• This must be packaged into a nucleus
that is about 5 micrometers in diameter
• This represents a compression of more
than 100,000!
• It is made possible by wrapping the DNA
around protein spools called nucleosomes
and then packing these in helical
filaments
Nucleosome Structure
• Chromatin, the nucleoprotein
complex, consists of histones and
nonhistone chromosomal proteins
• % major histone proteins: H1, H2A,
H2B, H3 and H4
• Histone octamers are major part of
the “protein spools”
• Nonhistone proteins are regulators
of gene expression
•4 major histone (H2A,
H2B, H3, H4) proteins
for octomer
•200 base pair long
DNA strand winds
around the octomer
•146 base pair DNA
“spacer separates
individual nucleosomes
•H1 protein involved in
higher-order chromatin
structure.
•W/O H1, Chromatin
looks like beads on
string
Solenoid Structure of Chromatin
RNA
• Single stranded molecule
• Chemically less stable than DNA
• presence of 2’-OH makes RNA more susceptible
to hydrolytic attack (especially form bases)
• Prone to degradation by Ribonucleases (Rnases)
• Has secondary structure. Can form intrachain
base pairing (i.e.cruciform structures).
• Multiple functions
Type of RNA
• Ribosomal RNA (rRNA) – integral part of
ribosomes (very abundant)
• Transfer RNA (tRNA) – carries activated
amino acids to ribosomes.
• Messenger RNA (mRNA) – endcodes
sequences of amino acids in proteins.
• Catalytic RNA (Ribozymes) – catalzye
cleavage of specific RNA species.
RNA can have extensive 2o
structure