Chapter 19 (part 2)
Nucleic Acids
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 and Nanotechnology
DNA and Nanotechnology
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