Transcript No Slide Title

Genes are composed of nucleic acids
(usually DNA)
• Pneumococcus can be transformed from an
avirulent to a virulent strain
• DNA is the transforming principle
• DNA in bacteriophage particles appears in
the progeny, but very little protein does.
Structures of nucleic acids
Nucleotides
DNA structures
Sedimentation and
Electrophoresis
A simple view of DNA
AGCCTCGCAT
TCGGAGCGTA
Nucleotides
• 3 components to nucleotides:
– Purine or pyrimidine base
– Ribose (RNA) or 2-deoxyribose (DNA)
sugar
– Phosphate
• Base + sugar = Nucleoside
• Base + sugar + phosphate = Nucleotide
Types of bases in nucleotides
Pyrimidine
O
O
CH3
HN
O
N
O
H
N
H
Thymine
Amino-
HN
Keto-
Uracil
Nucleotides: purine bases
NH 2
O
N
N
N
N
H
Adenine
6-aminopurine
N
HN
H2 N
N
N
H
Guanine
A keto-purine
Bases are attached to C1’ of the
sugar via an N-glycosidic bond
NH 2
N
N
N
N
HO
CH2
5'
O
1'
3'
OH
2’-deoxy- Adenosine , a nucleoside
Phosphate is attached to C5’ of the sugar
1st phosphate is a phosphoester, others are
attached as phosphoanhydrides.
O
=
O
=
NTP is
=
O
- O-P- O-P- O-P-O
O- O - O -
phosphoanhydride
O
OH OH
phosphoester
g
b a
base
Structure of a dinucleotide
The 3’ C of one
nucleotide is linked
to the 5’ C of the
next nucleotide in a
phosphodiester
linkage.
NH 2
N
O
O P O
O
N
5'
O
CH2
O
NH 2
N
3'
O H
O
P O
O
5' cytidylyladenylate
or
5'pCpA
N
5'
CH2
O
OH H
3'
N
N
Nucleic acids are linear chains of
nucleotides
• The 3’ C of one nucleotide is linked to the 5’
C of the next nucleotide.
• The linkage is by a phosphoester.
• The chain has an orientation defined by the
sugar-phosphage backbone.
• One terminal nucleotide has a “free” 5’ end,
and the other has a “free” 3’ end.
• Thus we designate orientation by 5’ to 3’.
More on orientation of chains of nucleic acids
• 5’ ACTG 3’ is different from 3’ ACTG 5’
• Unless specified otherwise, a chain is
written with the 5’ end on the left and the 3’
end on the right.
• When complementary strands in DNA are
written, usually the top strand is written 5’ to
3’, left to right, and the bottom strand is
written 3’ to 5’, left to right.
5’ GATTCGTACCG
3’ CTAAGCATGGC
Basics of DNA structure
• 2 complementary strands of nucleic acids
• Complementarity is based on H-bonding
between
– Keto bases with amino bases
– Pyrimidines with purines
•
•
•
•
A pairs with T (or U)
G pairs with C
The complementary strands are antiparallel.
The complementary strands are coiled
around each other.
Duplex DNA
•
•
•
•
•
•
Two strands coil around each other.
Right-handed coils (B form and A.
Coils form major and minor grooves.
Strands have opposite polarity (antiparallel).
Opposing bases in strands are complementary.
Different edges of paired bases are exposed in
major and minor grooves.
• Sugar-phosphate backbone is on the outside,
bases on the inside
– B-form DNA: base pairs are close to center of long axis
of the duplex.
– A-form nucleic acids: base pairs stack away from long
axis.
Implications of complementarity
• One chain (strand) of DNA can serve as the
template for synthesis of the
complementary chain.
• DNA replication: sequence of nucleotides
in one chain of the duplex determines the
sequence of nucleotides in the other chain.
• Transcription: sequence of nucleotides in
one chain of the duplex determines the
sequence of nucleotides in mRNA or its
precursor.
Base pairs in DNA
Major groove
Major groove
H
N
N
O
N
N
deox y ribos e
H
H N
H
N
N
N
N H
O
H
deox y ribos e
Minor groove
Guanine : Cytosine
N
N
deoxyribose
N
H
H
CH3
O
N
N
N
O
deoxyribose
Minor groove
Adenine : Thymine
Major types of duplex nucleic acid structures
• B form
– Most common form of DNA
– Right handed DNA-DNA helix
– Base pairs stack close to DNA central axis
• A form
– right handed RNA-DNA and RNA-RNA helix
– Base pairs stack away from the central axis
• Z form DNA
– Repeating purines and pyrimidines
– Left-handed helix
– May serve as some regulatory signal in cells
Forms of nucleic acid duplexes
B-form DNA
A-form
(e.g. duplex RNA)
Z DNA
Helical parameters for B, A and Z nucleic
acids
helix sense
bp per turn
vertical rise per bp
rotation per bp
helical diameter
B
RH
10
3.4
+36
19
A
RH
11
2.56
+33
23
Z
LH
12
3.7 Angstroms
-30 degrees
18 Angstroms
Hyperchromic shift when DNA is denatured
Native duplex DNA
denaturation by heat or
increasing pH
Denatured, strands are separate
+
renaturation by cooling or
lowering pH
hyperchromic
lower A
higher A
260
260
hypochromic
1.4
A
260
1.2
1.0
T = melting temperature, midpoint of the transition
m
Temperature
Factors that affect melting temperature, p. 85
• The melting temperature (Tm) increases as
– Increase G+C
– Increase ionic strength
(or m)
• Tm decreases as
– Increase denaturants
– Increase number of
mismatches
m  
m  
% G+C
Tm
Tm = 0.41 (% G+C) + 16.6 log M + 81.5 -0.7 (% formamide)
-1o (% mismatch)
Electrophoresis to measure SIZE
Size markers
DNA samples
-
+
For molecules of the same shape, log M is
inversely proportional to d.
For molecules of the same size, more compact
forms, such as supercoiled DNA, moves faster
than more extended forms, such as linear DNA.
Example of gel electrophoresis
Markers
Alpha-globin
gene
PCR product
217 bp
400 base pairs
300
200
100