Department of Chemistry and Biochemistry
University of Lethbridge
Biochemistry 3020
I. Biopolymers
Nucleic Acids
Nucleotides are the building blocks of nucleic
acids.
three characteristic components
(1) a nitrogen-containing base
(2) a pentose
(3) a phosphate
Bases are derived of two
parent compounds
Pyrimidine and Purine.
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Nitrogen-Containing Bases in Nucleic Acids
Two purine bases are commonly found in nucleic acids
But three pyrimidine bases
Several tautomers exist.
pH dependent
The Lactam form predominates at pH 7
Other forms become more prominent as pH decreases
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Some variations do occur.
Modified bases occur in DNA
In plants
In bacterial DNA
In bacterial DNA after
Bacteriophage infection
Some variations do occur.
Unusual bases in RNA
Commonly found in tRNAs
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Nucleic acids have two kinds of pentoses
The 5 carbon sugars can be:
deoxyribose
ribose
DNA
RNA
Is cytosine a base, a nucleoside or a nucleotide?
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Conformational freedom
The conformation of a nucleotide unit
is specified by seven torsion angles
The glycosidic bond:
1
β
α
The sugar-phosphate backbone: 1-6
χ
γ
δ
ε
Nucleotide
unit
ζ
Sugar ring pucker.
Planar form
C5’ defines
end or exo
Puckered form
C3’-endo conformation
half chair conformation
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Sugar ring pucker.
Known nucleotide structures show only few conformations
mostly C2’-endo
common C3’-endo and C3’-exo
Torsion about the glycosidic bond
The rotation of a base about its glycosidic bound is greatly hindered.
Purine residues exist in both syn and anti conformation.
Pyrimidines only in the anti conformation
In double helical Nucleic acids
All Bases are in the anti conformation
Exception Z-DNA
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Base pairing
Watson-Crick base pairing
two hydrogen bonds
three hydrogen bonds
Chargaffs rule (1950): A equals amount of T
and G equals amount of C
Base pairing
Watson-Crick base pairing
Major groove
T
A
Features of the Watsen-Crick base pair
The Watson-Crick base-pair is planar
Permitted H-bonds: A with T ( 2 bonds)
G with C ( 3 bonds)
Minor groove
C
Major groove
G
The dimensions of the base-pairs are similar
e.g. C1’ – C1’ distances
There is both hydrogen bonding and shape
complementarity in the base-pair
Minor groove
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Features of the Watson-Crick model of B-DNA
Antiparallel double helix
Right-handed helix
Base-pairs are perpendicular
to the axis of the helix
The axis of the helix passes
through the center of the
base pairs
Each base pair is rotated by
36° from the adjacent
base pair
The Base-pairs are stacked
0.34 nm from one another
Features of the Watson-Crick model of B-DNA
The Base-pairs are stacked
0.34 nm from one another
The double helix repeats
every 3.4 nm
34 Ǻ
B-DNA has two distinct
grooves: a MAJOR groove;
and a MINOR groove.
Minor
Major
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Forces responsible for the stability of B-DNA
Hydrophobic base stacking
Interactions (van der Waals forces)
between adjacent base pairs.
Hydrogen bonds
forming the base-pairs
34 Ǻ
Hydrogen bonds
due to the formation of a water
spine in the minor groove
Minor
Major
A-DNA and RNA
No water spine in A-DNA
Base-Pairs are pushed towards
the minor groove.
Axis no longer passes through
The center of each base-pair.
Base-Pairs tilt 19 degrees from
Perpendicular to the helix axis
34 Ǻ
Minor groove becomes as wide
As the major groove.
Major groove becomes very
deep but the minor groove is
very shallow.
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A-DNA and RNA
Back to sugar pucker:
In B-DNA, the C2’ atom lies above
the plane (C2’-endo),
while in A-DNA, the C3’ atom lies
above the plane (C3’-endo)
In RNA,
this distances the phosphate from the C2’ hydroxyl to prevent autocatalysis
Z-DNA forms a left-handed helix
Found in the Crystal structur
of the DNA molecule of the selfcomplementary hexanucleotide
CGCGCG
in 1979 by Alex Rich.
Left-handed double helix
12 Watson-Crick base-pairs
per turn.
A pitch of 44Ǻ
A deep minor groove but
almost no major groove.
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Z-DNA forms a left-handed helix
A deep minor groove but
almost no major groove.
Base-pairs are flipped 180°
compared to B-DNA
The repeating unit is a dinucleotide
d(XpYp)
Assumed by polynucleotides with
alternating purins and pyrimidines
Stabilized by high salt
(reduces electrostatic repulsion
between phosphate groups on
opposite strands)
Does Z-DNA exist in vivo?
Alex Rich discovered a family
of Z-DNA binding protein domains,
Zα s.
e.g. present in the RNA editing enzyme
ADAR1
Crystal Structure Of The Zα Z-DNA Complex, 1QBJ
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Summary of the DNA structures
Base pairing
Non Watson-Crick base pairing
Unconstrained A·T base pairs assume
Hoogsteen geometry.
T
T
A
Hooksteen geometry
N7 as the hydrogen bonding ecceptor
A
Watson-Crick geometry
N1 as the hydrogen bonding ecceptor
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Unusual DNA structures
Nucleotide participating in a Watson-Crick base pair can form
additional hydrogen bonds.
most stable at low pH
Triplex DNAs
Unusual DNA structures
Tetraplex
Occurse readily only
for DNA seqeunces
with a high proportion of G
G
G
G
G
Stable over a wide range of conditions
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The Structures of RNA
Single stranded RNA tends to assume a right-handed helical conformation
Structure is stabilized by base stacking
stronger between purine purine
than between pyrimidines
But
RNA has no simple, regular secondary structure, that serves as reference point.
Any self-complementarity will produce complex secondary structures.
Common secondary structure elements in RNA
RNA molecules fold to
Form local regions of A-form double helix
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Common secondary structure elements in RNA
RNA molecules fold to
Form local regions of A-form double helix
Common secondary structure elements in RNA
RNA molecules fold to
Form local regions of A-form double helix
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Common secondary structure elements in RNA
RNA molecules fold to
Form local regions of A-form double helix
Pseudoknots
RNA molecules fold to form complex
structures
Ten possible purine-pyrimidine
base pairs
Watson-Crick,
Reverse Watson-Crick,
Hoogsteen,
Reverse Hoogsteen,
Wobble
Reverse Wobble
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Predicting RNA structure
Cloverleaf secondary structure of tRNA
Predicting RNA structure
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Predicting RNA structure
Secondary structure :
MFOLD by Michael Zuker
http://www.bioinfo.rpi.edu/applications/mfold/old/rna/
Mfold web server for nucleic acid folding and
hybridization prediction.
Nucleic Acids Res. 31 (13), 3406-15, (2003)
Tertiary structure :
NO
RNAs can be very complex
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Ribozymes
Scissile bond
The hammerhead ribozyme
divalent cations are required
e.g. Mg2+ or Mn2+
PDB ID 1MME
SELEX
Systematic Evolution of Ligands by Exponential Enrichment
Is used to generate aptamers,
oligonucleotides, that bind to
a specific target.
Can be automated
Complete randomistaion of
25 nucleotides lead to:
25
4
= 1015
1. reverse transcriptase
2. RNA polymerase
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SELEX
Systematic Evolution of Ligands by Exponential Enrichment
Critical sequence features
AMP
Binds nucleotides (ATP and others)
KD < 50 µM
PDB ID 1RAW
Reading
Lehninger 4/e
Chapter 7
CARBOHYDRATES
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DNA Topology
E. Coli – 1 µm x 2 µm
E. Coli – 4.6 x 106 bp
B-DNA rise = 0.34 nm / bp
4.6 x 106 bp x 0.34
= 1,564,000 nm
= 1,56 mm
What to do?
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