MOLECULAR BIOLOGY – DNA structure, genetic code
DNA STRUCTURE, GENETIC CODE, CHROMOSOMES
MOLECULAR BIOLOGY – DNA structure, genetic code
GENES ARE ON CHROMOSOMES
DNA is the carrier of the
genetic information
MOLECULAR BIOLOGY – DNA structure, genetic code
DISCOVERY OF THE STRUCTURE
OF DNA
MOLECULAR BIOLOGY – DNA structure, genetic code
DNA STRUCTURE SHOULD FIT INTO 4 PRINCIPLES:
1. Provide a means for its own replication
2. Be able to encode the genetic information
3. Direct cell function
4. Accommodate changes caused by ‘mutations’
MOLECULAR BIOLOGY – DNA structure, genetic code
James D. Watson
15 years old accepted to University
22 years old received Ph.D. in zoology at Indiana
University, Bloomington
1950 breif research stay in Denmark and attended a
conference in Napoli - developing interest in DNA
Lecture by Maurice Wilkins
(Kings College of London: KCL)
- study of DNA structure
by X-ray diffraction of DNA crystals
Watson moved to Cambridge in order
to learn X-ray diffraction/
crystalography
MOLECULAR BIOLOGY – DNA structure, genetic code
X-ray crystalography
Diffracted Xrays
Photographic film
X-ray beam
X-ray source
‘crystalised’
sample
e.g. protein or
DNA fibre
Analysis of the diffracted X-rays detected on the photographic film yields
structural information about the crystalised sample
MOLECULAR BIOLOGY – DNA structure, genetic code
Cavendish Laboratory, Cambridge
Watson: studing the 3D-structure
of myoglobin (X-ray
crystalography)
Francis Crick
33 years old Ph.D. student (WWII)
studing haemoglobin - physics
background
Both men were interested in the
problem of how genetic
information was molecularly
stored - favouring DNA
Wanted to solve DNA structure
MOLECULAR BIOLOGY – DNA structure, genetic code
Chemical composition of DNA was known:
Erwin Chargaff (rules)
DNA of any species always had
equal concentrations of A & T
and G & C bases suggesting a
fixed relationship in DNA
DNA long polymer
Phosphate
Nitrogen containing bases
4 bases: Adenine, Guanine, (Purine
bases)
Thymine (T) and Cytosine
(Pyrimidine bases)
5-carbon sugar (2’-deoxyribose)
The molecular structure of these componenets was however unknown
MOLECULAR BIOLOGY – DNA structure, genetic code
Linus C. Pauling
3D structure of proteins by Xray crystalography
Keratin (William Astbury‘s
alpha form of protein - also a beta
form)
alpha-helix
Models of aminoacids
cut from paper
plausible 3-D models could be
built from knowledge of
chemical bonding and bond
distances to fit experimental
data.
Very eminent and respected molecular biologist who was known to be working on
uncovering the molecular structure of DNA
MOLECULAR BIOLOGY – DNA structure, genetic code
Rosalind E. Franklin
(1920-1958)
Colleague of Wilkins at KCL
(albeit a fractous one)
Exceptionally talented experimental chemist
with extensive experience in X-ray
crystalography
Discovered by careful experimentation and optimisation that DNA could
exists in a dehydrated ‘A-form’ and a fully hydrated ‘B-form’
MOLECULAR BIOLOGY – DNA structure, genetic code
NOVEMBER 1951 – Rosalind Franklin gave a seminar on her DNA
crystalography experiments
James Watson attended the seminar:
• driven by the perceived competition from Pauling, Watson & Crick proposed their
first model of the structure of DNA
• it was an embarrassing failure and was quickly discredited
• the model incorrectly placed the phosphate groups at the inside and bases on the
outside
• later emerged that Watson had incorrectly recalled Franklin’s data!
Franklin’s excellent background in physical chemistry and her
knowledge of the different hydration forms of DNA allowed her to
dispute that hydrophilic phosphate groups would be in the centre
whilst the hydrophobic bases would be on the outside!
MOLECULAR BIOLOGY – DNA structure, genetic code
Prior to Franklin‘s identification of ‘A’ and ‘B’ forms of DNA, complete interpretation of
DNA X-ray diffraction patterns was hampered by the presence of both hydration forms in
the crystal - (Wilkins and Astbury)
1952
Franklin (Ph.D. student Raymond Gosling) produced a very high
resolution X-ray diffraction image from a pure crystal of Bform DNA
‘Photo 51’
IMPORTANT DEDUCTIONS/ HINTS:
1) DNA was helical and most likely a double helix
consisting of 2 anti-parallel strands
2) Phosphates were on the outside of the helicies
with the bases on the inside
3) The distance between bases (3.4A), the length
of the period (34A i.e. 10 bases per turn of
helix) and the rise of the helix (36 degrees)
Franklin was characteristically cautious about over interpretation of the data
MOLECULAR BIOLOGY – DNA structure, genetic code
Early 1953
Gosling
Franklin about to leave
KCL was instructed that
the DNA work was to
remain in there! She was
preparing and had already
submitted manuscripts.
MRC
grant
report
Data (inc.
photo 51)
Wilkins
Wilkins showed Watson
‘photo 51’ without
permission of Franklin
Watson
Watson: "The instant I saw the picture
my mouth fell open and my pulse began to
race"
DOUBLE HELIX!
Max Perutz
Cavendish laboratory
Crick
Detailed calculations
suggested two strands
running in opposite
directions with bases
on inside
MOLECULAR BIOLOGY – DNA structure, genetic code
How are the two strands held together & how do the bases
interact with each other?
In 1952 Crick was speculating about the potential attractive
forces between the bases:
• mathematician friend John Griffith theorised which bases
were most likely to be attracted to each other based quantumn
mechanics
• Griffith suggested A-T and G-C as the most chemically
attractive combinations
• at the time Crick was unaware of Chargaffs rules!
Meanwhile Watson had been attempting to model base
interactions by ‘playing’ with cardboard cut outs (c.f. Linus Pauling
and the discovery of the protein alpha-helix)
Modelling proved unsuccessful
as hydrogen bonding between
base pair combinations seemed
too weak and unsatisfactory!
MOLECULAR BIOLOGY – DNA structure, genetic code
However bases can exist in two TAUTOMERIC FORMS
ENOL FORM
KETO FORMS
Jerry Donohue
Donohue advised Watson and Crick that the base tautomers in DNA are most
likely to be the Keto form and not the Enol form they had been modelling
MOLECULAR BIOLOGY – DNA structure, genetic code
Modelling using the keto form the hydrogen bonding worked!
Moreover the specific base-pair combinations agreed
with both Griffith’s theory and Chargaff’s rules
MOLECULAR BIOLOGY – DNA structure, genetic code
James Watson and Francis Crick now
had all the information they needed to
build their model and publish the
molecular structure of DNA
MOLECULAR BIOLOGY – DNA structure, genetic code
DNA STRUCTURE SOLVED!
Nature April 25, 1953.
(immortality without even one experiment of their own!)
MOLECULAR BIOLOGY – DNA structure, genetic code
DEOXYRIBONUCLEIC ACID - DNA
A-T & G-C hydrogen
bonding base pairs
Phosphate
Nitrogen containing base
4 bases: adenine, guanine, thymine, cytosine
5-carbon sugar
Phosphodieste
r backbone
MOLECULAR BIOLOGY – DNA structure, genetic code
The two DNA strands have directionality as they are polarized
polymers that run anti-parallel to each other
3’ OH
5’ P
The repeating unit of the DNA polymer is the nucleotide (either; A, T, G
or C), that is based around the 5 carbon sugar deoxyribose
Each carbon in the
deoxyribose sugar is
numbered with 1’ - 5’
nomencluture
5’5’
4’
3’3’
1’
2’
DNA polymer formed by the formation of
phosphodiester bonds between the 5’
phosphate group and the 3’ hyrdroxl
group
deoxyribose
Therefore one end of each strand contains a 5’ phosphate group (actually
triphosphate) whilst the other end contains 3’ hydroxl group
5’ P
3’ OH
MOLECULAR BIOLOGY – DNA structure, genetic code
1962 Nobel Prize for medicine: Francis Crick, James Watson and Maurice Wilkin
Rosalind E. Franklin
1958 (37 years)
MOLECULAR BIOLOGY – DNA structure, genetic code
DNA STRUCTURE SHOULD FIT INTO 4 PRINCIPLES:
1. Provide a means for its own replication
2. Be able to encode the genetic information
3. Direct cell function
4. Accommodate changes caused by ‘mutations’
MOLECULAR BIOLOGY – DNA structure, genetic code
Watson & Crick knew that their
DNA structure provided a
possible copying mechanism
based on specifc base-pairing
How could this be achieved?
MOLECULAR BIOLOGY – DNA structure, genetic code
DNA replication theories
- proposed by Watson and Crick
How do we experimentally test these theories?
MOLECULAR BIOLOGY – DNA structure, genetic code
DNA replication: Meselson-Stahl experiment
• DNA extracted from E-coli grown for many generations on a heavy
specifically sediment in a salt gradient
15N
isotope of nitrogen will
• by following the sedimentation characteristics of DNA extracted from E-coli transferred back to
normal 14N containing media one can infer the mechanism of DNA replication after each cell division
heavy isotope 15N
possible replication mechanisms
normal 14N
cell generation
The DNA must replicate in a semi-conservative fashion as predicted by Watson & Crick
(expanded upon on later lectures)
MOLECULAR BIOLOGY – DNA structure, genetic code
Meselson-Stahl experiment video/ tutorial
http://www.sumanasinc.com/webcontent/animations/content/meselson.html
MOLECULAR BIOLOGY – DNA structure, genetic code
DNA STRUCTURE SHOULD FIT INTO 4 PRINCIPLES:
1. Provide a means for its own replication
2. Be able to encode the genetic information
3. Direct cell function
4. Accommodate changes caused by ‘mutations’
MOLECULAR BIOLOGY – DNA structure, genetic code
INFORMATION
The Central Dogma of Molecular Biology - Francis Crick 1958
Details in later
lectures
The genetic flow of information in a cell starts with DNA ‘instructions’ and passes through
RNA ‘intermediates’ that dictate the synthesis of ‘functional’ protein
BUT WHAT IS THE CODE BEHIND THIS TRANSFER OF GENETIC INFORMATION ?
MOLECULAR BIOLOGY – DNA structure, genetic code
Cracking the Genetic Code
Proteins consist of 20 different amino acids whereas DNA/ RNA have only 4 different
nucleotides (Uracil, replacing T in RNA):
If a sequence of 2 nucleotides encoded a single amino acid the code could only accommodate
16 amino acids (i.e. 42)
however a triplet nucleotide could code for potentially up to 64 amino acids (43)
Marshall Nirenberg
& Heinrich Matthaei
Synthetic poly-uracil RNA
+ 1 radiolabelled amino acid
+ 19 unlabelled amino acids
Observe if the radioactively
labelled amino acid would be
incorporated into protein?
Only when using labelled phenylalanine did the
poly-uracil RNA lead to the production of
radioactive protein
Lysed E-coli cell lysate (protein synthesis
apparatus intact)
The genetic code for the incorporation of
phenylalanine into proteins had been cracked
MOLECULAR BIOLOGY – DNA structure, genetic code
The Genetic Code
N.B. that the
genetic code is
largely redundant
with most amino
acids having
more than one
codon
Three codons do
not lead to
incorporation of
any amino acids
- play role in
terminating
protein synthesis
Methionine and
tryptophan only
have one codon
Similar experiments identified the other three letter ‘codons’ found in messenger
RNAs (mRNAs) responsible for the incorporation of the remaining amino acids
into protein - Nobel Prize of 1968
MOLECULAR BIOLOGY – DNA structure, genetic code
Cracking the genetic code video/ tutorial
http://bcs.whfreeman.com/thelifewire/content/chp12/1202002.html
MOLECULAR BIOLOGY – DNA structure, genetic code
DNA sequence driven Genetic Code of the Central Dogma
double stranded DNA
5’
3’
ATG GCT CCT TCT TCC AGA GGT GGC . . . . . . TAA
TAC CGA GGA AGA AGG TCT CCA CCG . . . . . . ATT
3’
5’
TRANSCRIPTION
single stranded mRNA
TRANSLATION
AUG GCU CCU UCU UCC AGA GGU GGC . . . . . . UAA
AUG
UAA
protein coding sequence
or
open reading frame
MAPSSRGG…..
Functional Protein
THE SEQUENCE OF SPECIFC NUCLEOTIDES IN DNA
DICTATES THE SEQUENCE OF AMINO ACIDS IN THE
FUNCTIONAL PROTEINS e.g. enzymes
MOLECULAR BIOLOGY – DNA structure, genetic code
DNA STRUCTURE SHOULD FIT INTO 4 PRINCIPLES:
1. Provide a means for its own replication
2. Be able to encode the genetic information
3. Direct cell function
4. Accommodate changes caused by ‘mutations’
MOLECULAR BIOLOGY – DNA structure, genetic code
Mutations are variations in the DNA sequence e.g. single base
pair substitution
double stranded DNA
5’
3’
TCT TCC AGA GGT GGC . . . . . . TAA
ATG GCT CCT TCA
AGA AGG TCT CCA CCG . . . . . . ATT
TAC CGA GGA AGT
3’
5’
TRANSCRIPTION
single stranded mRNA
TRANSLATION
AUG GCU CCU UCU
UCA UCC AGA GGU GGC . . . . . . UAA
AUG
UAA
protein coding sequence
or
open reading frame
MAPSSRGG…..
MAPSSSGG…..
Functional Protein
SUCH MUTATIONS CAN INFLUENCE THE
FUNCTIONALITY OF THE PROTEIN e.g. changing
which amino acid is incorporated
MOLECULAR BIOLOGY – DNA structure, genetic code
Most DNA is not coding for proteins !
Only 1.5% of the human DNA genome directly encodes amino acids for
incorporation into proteins
MOLECULAR BIOLOGY – DNA structure, genetic code
How is DNA organised in the cell?
600x
Human DNA:
3 200 000 000 letters
200x 500-pages books
A single cells stretched out DNA = 1.8m
MOLECULAR BIOLOGY – DNA structure, genetic code
Bacterial DNA ~ 1 mm long
Most bacterial DNA exists in a covalently
closed circular form
1000 x more than
~ 1 mm
SUPERCOILING
…thanks to mobiles
no more twisted telephone cords!
MOLECULAR BIOLOGY – DNA structure, genetic code
TOPOISOMERASES – enzymes that insert or remove supercoils
Type I … break only one strand -> relaxing or twisting of the helix
Type II … break both strands and pass another part of the double
helix through the gap
protein
scaffold
A typical bacterial chromosome
consists of about 50 giant
supercoiled loops of DNA
MOLECULAR BIOLOGY – DNA structure, genetic code
Eukaryotic DNA is complexed with HISTONE proteins that together form
more and more ordered structures of CHROMATIN resulting in
chromosomes
Nucleosome
H2A, H2B,
H3, H4
80 bp
80 bp
200 bp
40 bp
MOLECULAR BIOLOGY – DNA structure, genetic code
Eukaryotic chromatin hierarcheal structure
‘beads on a
string’
30nm ‘solenoid
fibre’
scaffold associated
fibres
Condensed
chromosome
Net result is that a eukaryotic (human) cell’s DNA is packaged into a mitotic chromosome
10,000 fold shorter than it extended length!
MOLECULAR BIOLOGY – DNA structure, genetic code
SECOND SUMMARY – CRUCIAL KNOWLEDGE
MOLECULAR BIOLOGY – DNA structure, genetic code
RNA
T A C C G T T A G T T C A C G A T T
A U G G C A A U C A A G U G C U A A
A T G G C A A T C A A G T G C . . .
. . . T A A
part of the chromosome
where gene X is located
STOP
START
CODING SEQUENCE
double-strand
DNA
TRANSCRIPTION
DNA  RNA
RNA
A U G G C A A U C A A G U G C U A A
TRANSLATION
Ribosomes, tRNAs (expanded later)
Met
Ala
Ala
Lys Ile
PROPERLY FOLDED PROTEIN
executes its function in cell
The Central Dogma
densely packed chromosome