Nucleotides and Nucleic Acids

Timeline
1800’s F Miescher - nucleic acids

1928 F. Griffith - Transforming principle

1944
Avery, McCleod & McCarty- Transforming
principle is DNA
1952 Hershey-Chase ‘blender’ experiment
1952 Erwin Chargaff – base ratios
1952 R Franklin & M Wilkins–DNA diffraction pattern

1953 J Watson and F Crick – DNA structure solved

 Nucleotides
•  “Energy rich” compounds
–  Chemical signals
–  Enzyme co-factors

•  Nucleic Acids
–  DNA and RNA
–  Polymers of nucleotides

•  3 components
–  Nitrogenous “base”
–  Ribose (or deoxyribose)
–  Phosphate
 “Bases”
• 2 purine bases
• Adenine: A A G
• Guanine: G

• Bases
• Pyrimidines
• Purines
C T U

• 2 pyrimidine bases (in DNA)

• Cytosine: C
• Thymine: T
• or Uracil: U
(in RNA, instead of Thymine)
 Ribose
•  carbons numbered:
–  1’,2’,3’,4’,5’
•  DNA:
–  2’ Deoxyribose
–  or just deoxyribose
5’

1’
4’

3’ 2’
 Nucleotides

Summary
DNA A,C,G,T
deoxyribose
RNA A,C,G,U
ribose
 Polymerise Nucleotides
N-glycosidic bonds

•  nucleotides can be linked

–  phosphates linked to 2 pentoses
–  phosphodiester linkages
•  Link PO4 at
–  5’ end to 3’ OH of next nucleotide

•  chain has POLARITY

– distinct ends
• 5’ end
• 3’ end
– usually “read” 5’ -> 3’
 •  The PO4 group of one
nucleotide is attached to
the sugar of the next
nucleotide in line.

•  The result is a
“backbone” of
alternating phosphates
and sugars, from which
the bases starts.

Fig. 16.3, Page 290

 Nucleotides as Energy Carriers

•  ATP
–  Adenosine triphosphate
•  ADP, AMP
–  Adenosine diphosphate
–  Adenosine monophosphate

ATP <--> ADP + PO4

ADP <--> AMP + PO4
•  Main energy exchange reactions in cells
 Structure of DNA?
•  The Genetic Material
•  Crick and Watson
–  Race with Linus Pauling to predict structure

•  Chargaff’s rules:
–  Chemical analysis:
[A] = [T]
[G] = [C]
–  Constant
•  % GC for each organism
–  over time
–  across all tissues
 X-Ray Diffraction
•  Predict
–  Double helix
–  2 periodicities
•  3.4Å
•  34Å
 Base Pairing

•  A – T basepair
–  2 h-bonds
•  G – C basepair
–  3 h-bonds

•  2 anti-parallel
DNA strands
 The Double Helix
•  3.4Å per basepair
•  10 basepairs per turn
–  10-11 in aqueous solution
•  2 anti-parallel strands
 Essential features of B-DNA
•  Right twisting
•  Double stranded
helix
•  Anti-parallel
•  Bases on the
inside
(Perpendicular to
axis)
•  Uniform diameter
(~20A)
•  Major and minor
groove
•  Complementary
base pairing
Why DNA evolved as the genetic material
but not RNA?

Maybe because RNA but not DNA is prone to base-

catalysed hydrolysis
 Stability of DNA

DNA must be stable in order to store genetic information.

What accounts for DNA’s stability?

Aromatic Stacking
Weak noncovalent force caused by overlapping of p-orbitals of the
bases in the nucleotide; also called pi stacking.

Hydrogen Bonding
- Millions of hydrogen bonds in DNA is the main structural feature
that explains why DNA is stable. Hydrogen bonding is strong, but can
easily be broken for DNA replication.
 DNA conformations
B-DNA:
–  right-handed double helix with a wide and narrow
groove.
A-DNA
–  major groove is very deep and the minor groove is
quite shallow
Z-DNA
–  consists of dinucleotides, each with different
conformations
4 stranded DNA
–  Telomeric DNA
 DNA conformations
A DNA Both form right-handed double helices
B-DNA helix has a larger pitch and
B DNA
hence a smaller width than that of A

In B-DNA, the helix axis passes through

the base pairs and hence B-DNA has no
internal spaces, whereas that of A-DNA
has a 6 Angstrom diameter hole along
its helical axis.

The planes of the base pairs in B-DNA

are nearly perpendicular to the helix
axis, whereas in A-DNA, they are
inclined from this. Therefore, B-DNA
has a wide and deep major groove and
a narrow and deep minor groove,
whereas A-DNA has a narrow and deep
major groove, but a wide and shallow
minor groove.
 DNA conformations
Z DNA B-DNA forms a right-handed
d o u b l e h e l i x i n w h i ch t h e
B DNA
repeating unit is a nucleotide,
whereas Z-DNA forms a left-
handed double helix in which the
repeating unit is a dinucleotide.

The Z-DNA helix has a larger

pitch and is therefore narrower
than that of B-DNA.

B-DNA has a wide and deep

major groove and a narrow and
deep minor groove, whereas Z-
DNA has a narrow and deep
minor groove but a nonexistent
major groove.
Table 27-1 frp,, 5th
 Human genome project
Goal: to sequence the entire human nuclear genome
Public consortium Celera Genomics
Headed by F Collins Headed by C Venter
Started in mid 80’s Started in mid 90’s
Working draft completed Working draft
in 2001 completed in 2001
Final sequence 2003
Nature: Feb 2001 Science: Feb 2001

Human genome = 3.3 X 109 base pairs

Number of genes = 26 – 32,000 genes
 Genetic material may be DNA
Double stranded DNA Single stranded DNA
linear linear

human chromosomes adeno-associated viruses

circular Prokaryotes circular

Mitochondria
Chloroplasts
Some viruses
(pox viruses)
Parvovirus
 Genetic material may be RNA
Double stranded RNA Single stranded RNA

Retroviruses like HIV

reoviruses
 RNA
•  Usually single stranded
•  Genetic material of RNA virus
•  Functional:
–  e.g. Translation machinery
•  rRNA (ribosomal RNA)
•  tRNA (transfer RNA)
•  Regulatory:
–  Control of gene expression
•  miRNA (microRNA)
•  Gene Expression
–  mRNA (messenger RNA)
–  Copy of 1 gene for translation by ribosomes
 EVOLUTION: Started
with an RNA world?

RNA enzyme
(ribozyme)

Protein DNA
 Types of cellular RNA
molecules

•  All RNA in E. coli (a bacterium) is made by one RNA polymerase

•  mRNA encodes protein amino acid sequences
 RNA Secondary Structure
•  RNA single stranded
•  Can form base pairs internally
 RNA Secondary Structure
•  Many functional RNAs
have secondary structure
•  G-U basepairs allowed
 Messenger RNA (mRNA)

This class of RNAs are the genetic

coding templates used by the
translational machinery to
determine the order of amino acids
incorporated into an elongating
polypeptide in the process of
translation.
 Transfer RNA (tRNA)
class of small RNAs
form covalent bonds to amino acids
allows correct insertion of amino acids
into the elongating polypeptide
chain.
 Ribosomes
Made of rRNA & ribosomal
proteins E.coli

2 subunits – large and small

Subunits are self assembling

combine only in the presence of

mRNA and a charged eukaryote

(aminoacylated) tRNA
 Ribosomes

Fig 4-24 from MCB by Lodish et al

 3) Ribosomes
Ribosomal RNA (rRNA) assembled,
together with numerous ribosomal
proteins, to form the ribosomes.

Ribosomes engage the mRNAs and form a

catalytic domain into which the tRNAs
enter with their attached amino acids. The
proteins of the ribosomes catalyze all of
the functions of polypeptide synthesis
 DNA RNA
CH2 o CH2 o
H H
H H H
H H
H H
OH
Deoxyribose Ribose
sugar (O on C2 sugar (no
is missed) missed O)
Deoxiribo-Nucleic-Acid Ribo-Nucleic-Acid

Double stranded nucleic acid Single stranded nucleic acid

Bases: A, G, C, T Bases: A, G, C, U
Repeated Sugar - Phosphate DNA backbone
Sugar–Phosphate-Base One nucleotide
Polynucleotide
DNA Molecule

DNA Double stranded

RNA single stranded

DNA A G C T A T C

mRNA T
U C G A T
U A G
 Properties of DNA

C-value paradox
Gene size, genome size
Topology of DNA
Denaturation and renaturation of DNA
 gene sizes
Largest known mammalian gene is
DMD gene 2.5 Mbp (0.1% of the genome)
Causes Duchenne’s muscular dystrophy (DMD)
characterized by rapid progression of muscle
degeneration which occurs early in life.

‘scoliosis’
 genome sizes
organism Number of base pairs (kb)
viruses
Lambda bacteriophage ( λ) 48.6
bacteria
Eschericia coli 4,640
eukaryotes
Yeast 13,500
Drosophila 165,000
Human 3.3 x 106
 Does size matter? C-value paradox
Boa constrictor
Genome size: 2.1 Gbp

Homo sapiens sapiens

Genome size: 3.2 Gbp

mountain grasshopper
Podisma pedestris
Genome size: 18 Gbp

protozoan
Amoeba dubia
Genome size: 670Gbp
C value: DNA content of a haploid cell
Comparative
genome sizes

C-value
paradox
Why is there a
discrepancy
between
genome size and
genetic
complexity?
Due to the presence
of repetitive (junk?)
DNA

Repetitive DNA
families constitute
nearly one-half of
genome (~45%)

Protein domains contribute to

organism complexity
 Topology of DNA

DNA supercoiling: coiling of a coil

Important feature in all chromosomes

Allows packing / unpacking of DNA

Supercoiled DNA moves faster than relaxed DNA

 Supercoiling topology

No supercoiling (left) to tightly supercoiled (right)

negatively supercoiled (right handed)
Results from under or unwinding
• 

Important in DNA packing/unpacking e.g during

• 
replication/transcription
positively supercoiled (left handed)
Results from overwinding
•  Analogy = phone cord
Also packs DNA but difficult to unwind
• 
Supercoiling takes 2 forms
toroidal (DNA around histones) or
interwound (bacterial chromosomes)
Supercoiled DNA needs to be
unwound /wound when necessary
Unwound during transcription/replication
Wound before cell division

Supercoils can be relaxed by

q Single strand nicks (topoisomerase I)
q Double strand breaks (toposiomerase II)
 Topoisomerase I
Cause temporary single
strand breaks in DNA
Allows free rotation of
helix
Acts by breaking
phosphodiester linkage
via tyrosine active site
Topoisomerase II (DNA gyrase)
ATP hydrolysis mediated double strand
break
Inhibitors are effective antibiotics and
cancer chemotherapy agents
Why does a plasmid (circular DNA) that has never been
cut give more than one band on a gel?

Full length linear

Relaxed circle

supercoiled

EBr
 Denaturation of DNA
Also called melting
Occurs abruptly at
certain
temperatures

Tm – temp at which
half the helical
structure is lost
DNA melting curve
 Tm varies according to the GC content
High GC content
- high Tm

GC rich regions
tend to be gene
rich
 Renaturation of DNA
Also called
annealing

Occurs ~ 25oC
below Tm

Property used
in PCR and
hybridisation
techniques