Nucleotides and Nucleic Acids -
Lehninger Chapter8
– Basics
– Structure
– Chemistry
– Nucleotide Function
1 Basics
–Building Blocks
–Canonical and Minor Bases
–Phosphodiester bonds
–Naming and Drawing
–Base Stacking and Pairing
Building Blocks
– Nucleotides = Base + Sugar + Phosphate
– Nucleosides = Base + Sugar
– Nitrogen Bases
• Purines (5 + 6 membered rings) – numbering
– Adenine Guanine
• Pyrimidines (6 membered ring) – numbering
– Thymine Cytosine Uracil
– Pentose Sugars (numbering)
•– Ribose
•– Deoxy Ribose
Ribosa dan
deoksiriboa
Ribose
Sugar “Pucker”
Canonical and Minor Bases
–DNA A, G, C, T
–RNA A, G, C, U
–Modified bases
–Methylation in DNA
–Lots of Mods in RNA
Purines
Phosphodiester bonds
– Formed by Polymerase and Ligase activities
– C-5' OH carries the phosphate in nucleotides
– C5' - O - P - O - C3'
– Phosphate pKa ~ 0
– Natural Oligonucleotides have 5' P and 3' 0H
– Base hydrolysis due to ionizaiton of 2' OH in
RNA
Oligonucleotide naming /
drawing conventions
– 5’ - Left to Right - 3’
– pACGTOH
– ACGT
Base Stacking and Base Pairing
– Bases are very nearly planar
– Aromaticity => large absorbance at 260nm
– Epsilon 260 ≈ 10,000 (M-1 cm-1 )
– The A260 ≈ 50 μg /ml for DS DNA
– The A260 ≈ 40 μg /ml for SS DNA or RNA
– Flat surfaces are hydrophobic
– Dipole-Dipole and Van Der Waals interactions also
stabilize stacked structures
– Bases have hydrogen bond donors and acceptors
– H-bonding potential satisfied in paired structures
2 Structure
– DNA contains genetic Information
– Distinctive base composition foretells base
pairing patterns
– Double helical structures
– Local structures
– mRNAs - little structure
– Stable RNAs - complex structures
DNA contains genetic
Information
– Purified DNA can "transform" Bacteria
• Avery, MacLeod & McCarty transferred the
virulence trait to pneumococci
– The genetic material contains 32P (DNA) and
not 35S (protein – C, M)
• Hershey and Chase grew bacteriophage on either 32P
or 35S
• Bacteriophage infection resulted in transfer of 32 P
and not 35S
Distinctive Base composition
foretell base pairing patterns
– Hydrolysis of DNA and analysis of base
composition
• Same for different individuals of a given species
• Same over time
• Same in different tissues
• %A = %T and %G = %C (Chargaff's Rules)
– Amino acid compositions vary under all three
conditions
– No quantitative relationships in AA composition
Structural Basis of
Chargaff’s Rules
• Two Strands have
complementary sequences
• 2 logical operations to
obtain complementary
strand 5' to 3'
– 1. Reverse: Rewrite the
sequence, back to front
– 2. Complement: Swap A with
T, C with G
Double helical structures
– Potentially Right or Left Handed
• Actually Mostly Right Handed
– Potentially Parallel or Anti-parallel
• Actually anti-parallel
– Sugar Pucker + 6 rotatable bonds gives 3 families
– A, B, Z structures
• http://www.olemiss.edu/depts/chemistry/courses/chem47
1_10/ABZ_DNA.kin
– KING 3D display software:
• http://kinemage.biochem.duke.edu/software/king.php
B-DNA
Semi-
conservative
Replication
DNA
Backbone
Flexibility
• Multiple
Degrees of
Rotational
Freedom
Glycosidic Bond Configurations
3 Canonical Helical DNA Structures
A, B and Z DNA
• A form – favored by RNA
• B form – Standard DNA
double helix under
physiological conditions
• Z form – laboratory
anomaly,
– Left Handed
– Requires Alt. GC
– High Salt/ Charge
neutralization
A, B & Z DNA Kinemages
Local structures
• Palindromes – Inverted repeats
– Not quite the same as (Madam I’m Adam)
– Symmetrical Sequence Elements Match
Symmetry of Protein Homo-Oligomers
– Symmetry often incomplete/imperfect
• Direct Repeats
• Hairpin and Cruciform Structures
Hoogstein Pairing in Base Triples
Messenger RNAs
• Contain protein coding information
– ATG start codon to UAA, UAG, UGA Stop Codon
– A cistron is the unit of RNA that encodes one
polypeptide chain
– Prokaryotic mRNAs are poly-cistronic
– Eukaryotic mRNAs are mono-cistronic
• Base pairing/3D structure is the exception
– Can be used to regulate RNA stability termination,
RNA editng, RNA splicing
The Genetic Code
G A C U
G gly arg ser arg trp cys
A glu asp lys asn gln his tyr
C ala thr pro ser
me
U val t ile leu leu phe
G A C U G A C U G A C U G A C U
• GG[GACU] code for Glycine
• UGG codes for Tryptophan
• UGA, UAG, UAA are stop codons
• AG[CU] and UC[GACU] code for Serine
mRNA coding patterns
Stable
RNAs with
complex
structures
RNA Helices are short, bulges,
loops
RNA Secondary
Structure Maps
• Calculated from
helix
thermodynamic
parameters
• Loop entropy
considerations
tRNA-Phe
2° Structure
tRNA - the prototype structure
tRNA Phe Kinemage
• http://www.olemi
ss.edu/depts/chem
istry/courses/che
m471/6tna.kin
3 Chemistry
– Denaturation and reannealing
– Hybridization
– Spontaneous Chemical Reactions
– Methylation
– Sequencing
– Chemical Synthesis
Denaturation and reannealing
Tm (transition midpoint) as a
function of base composition
• Salt
dependence is
more dramatic
Hybridization
• DNA sequences can
spontaneously re-
anneal and form
helices
• Basis for many of
molecular biology
techniques.
– PCR, DNA
sequencing
Spontaneous
Chemical Reactions
Pyrimidine Dimers
Keto – Enol Tautomerization
Methylation Reactions
DNA Polymerase
Mechanism
Di-deoxy NTPs – Chain
terminators
Sanger
Sequencing
Dye-Terminator (one-tube rxns)
Next-generation
DNA sequencing
• Millions of DNA
fragments isolated,
amplified by PCR and
monitored in parallel
• Jay Shendure & Hanlee Ji
• Nature Biotechnology 26, 1135 -
1145 (2008)
• Published online: 9 October 2008
doi:10.1038/nbt1486
DNA Solid-Phase
Synthesis
4 Nucleotide Function
– Energetic Intermediates
– Adenine Enzyme Cofactors
– Regulatory Molecules
Nucleotides
Phospho-Anhydrides and Phosphate
Esters – High Energy Bonds
Co-Enzyme A – Carrier for
Acetyl units in intermediary
metabolism, fatty acid synthesis
and oxidation
NAD+/NADH, NADP+/NADPH
and FAD/FADH2
• Redox Cofactors
Regulatory/Signalling Molecules
