DNA STRUCTURE, GENETIC CODE, CHROMOSOMES
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Transcript of DNA STRUCTURE, GENETIC CODE, CHROMOSOMES
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
MOLECULAR BIOLOGY – DNA structure, genetic code
DISCOVERY OF THE STRUCTUREOF DNA
DISCOVERY OF THE STRUCTUREOF DNA
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 University22 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
MOLECULAR BIOLOGY – DNA structure, genetic code
Watson moved to Cambridge in order to learn X-ray diffraction/
crystalography
X-ray crystalography
MOLECULAR BIOLOGY – DNA structure, genetic code
‘crystalised’ sample
e.g. protein or DNA fibre
X-ray source
X-ray beam
Diffracted X-rays
Photographic film
Analysis of the diffracted X-rays detected on the photographic film yields structural information about the crystalised sample
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
MOLECULAR BIOLOGY – DNA structure, genetic code
Both men were interested in the problem of how genetic
information was molecularly stored - favouring DNA
Wanted to solve DNA structure
Chemical composition of DNA was known:
5-carbon sugar (2’-deoxyribose)
Nitrogen containing bases
Phosphate
4 bases: Adenine, Guanine, (Purine bases) Thymine (T) and Cytosine (Pyrimidine bases)
DNA long polymer
MOLECULAR BIOLOGY – DNA structure, genetic code
Erwin Chargaff (rules)
DNA of any species always had equal concentrations of A & T and G & C bases suggesting a fixed relationship in DNA
The molecular structure of these componenets was however unknown
Linus C. Pauling
Models of aminoacidscut from paper
plausible 3-D models could be built from knowledge of
chemical bonding and bond distances to fit experimental
data.
Keratin (William Astbury‘s alpha form of protein - also a beta form)
alpha-helix
MOLECULAR BIOLOGY – DNA structure, genetic code
Very eminent and respected molecular biologist who was known to be working on uncovering the molecular structure of DNA
3D structure of proteins by X-ray crystalography
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
MOLECULAR BIOLOGY – DNA structure, genetic code
Discovered by careful experimentation and optimisation that DNA could exists in a dehydrated ‘A-form’ and a fully hydrated ‘B-form’
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
Franklin was characteristically cautious about over interpretation of the
data
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)
MOLECULAR BIOLOGY – DNA structure, genetic code
1952Franklin (Ph.D. student Raymond Gosling) produced a very high resolution X-ray diffraction image from a pure crystal of B-form 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)
MOLECULAR BIOLOGY – DNA structure, genetic code
Early 1953
Watson: "The instant I saw the picture my mouth fell open and my pulse began to race"
DOUBLE HELIX!
Detailed calculations suggested two strands
running in opposite directions with bases
on inside
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)
Watson
Wilkins showed Watson ‘photo 51’ without
permission of Franklin
Wilkins
Gosling
Max Perutz Crick
Cavendish laboratory
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:
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)
MOLECULAR BIOLOGY – DNA structure, genetic code
• 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!
Modelling proved unsuccessful as hydrogen bonding between
base pair combinations seemed too weak and unsatisfactory!
ENOL FORM KETO FORMS
However bases can exist in two TAUTOMERIC FORMS
Jerry Donohue
MOLECULAR BIOLOGY – DNA structure, genetic code
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
DNA STRUCTURE SOLVED!
(immortality without even one experiment of their own!)
MOLECULAR BIOLOGY – DNA structure, genetic code
Nature April 25, 1953.
5-carbon sugar
Nitrogen containing base
Phosphate
4 bases: adenine, guanine, thymine, cytosine
DEOXYRIBONUCLEIC ACID - DNA
MOLECULAR BIOLOGY – DNA structure, genetic code
Phosphodiester backbone
A-T & G-C hydrogen bonding base pairs
The two DNA strands have directionality as they are polarized polymers that run anti-parallel to each other
MOLECULAR BIOLOGY – DNA structure, genetic code
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
1’
2’3’
4’
5’
DNA polymer formed by the formation of phosphodiester bonds between the 5’ phosphate group and the 3’ hyrdroxl
group
5’
3’
Therefore one end of each strand contains a 5’ phosphate group (actually triphosphate) whilst the other end contains 3’ hydroxl group
3’ OH
3’ OH
5’ P
5’ P
deoxyribose
1962 Nobel Prize for medicine: Francis Crick, James Watson and Maurice Wilkins
1958 (37 years)
Rosalind E. Franklin
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
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?
DNA replication theories
MOLECULAR BIOLOGY – DNA structure, genetic code
How do we experimentally test these theories?
- proposed by Watson and Crick
DNA replication: Meselson-Stahl experiment
normal 14N
MOLECULAR BIOLOGY – DNA structure, genetic code
• DNA extracted from E-coli grown for many generations on a heavy 15N isotope of nitrogen will specifically sediment in a salt gradient
heavy isotope 15N
cell generation
• 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
possible replication mechanisms
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
http://www.sumanasinc.com/webcontent/animations/content/meselson.html
Meselson-Stahl experiment video/ tutorial
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
MOLECULAR BIOLOGY – DNA structure, genetic code
The Central Dogma of Molecular Biology - Francis Crick 1958
The genetic flow of information in a cell starts with DNA ‘instructions’ and passes through RNA ‘intermediates’ that dictate the synthesis of ‘functional’ protein
INF
OR
MA
TIO
N
Details in later lectures
BUT WHAT IS THE CODE BEHIND THIS TRANSFER OF GENETIC INFORMATION ?
Marshall Nirenberg &
Heinrich Matthaei
Cracking the Genetic Code MOLECULAR BIOLOGY – DNA structure, 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)
Lysed E-coli cell lysate (protein synthesis apparatus intact)
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
The genetic code for the incorporation of phenylalanine into proteins had been cracked
MOLECULAR BIOLOGY – DNA structure, genetic code
The Genetic Code
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
N.B. that the genetic code is
largely redundant with most amino
acids having more than one
codon
Methionine and tryptophan only have one codon
Three codons do not lead to
incorporation of any amino acids
- play role in terminating
protein synthesis
MOLECULAR BIOLOGY – DNA structure, genetic code
Cracking the genetic code video/ tutorial
http://bcs.whfreeman.com/thelifewire/content/chp12/1202002.html
AUG UAA
double stranded DNA
TRANSLATIONprotein coding sequence
or open reading frame
Functional Protein
MAPSSRGG…..
MOLECULAR BIOLOGY – DNA structure, genetic code
DNA sequence driven Genetic Code of the Central Dogma
ATG GCT CCT TCT TCC AGA GGT GGC . . . . . . TAATAC CGA GGA AGA AGG TCT CCA CCG . . . . . . ATT
5’5’3’
3’
single stranded mRNA AUG GCU CCU UCU UCC AGA GGU GGC . . . . . . UAA
TRANSCRIPTION
THE SEQUENCE OF SPECIFC NUCLEOTIDES IN DNA DICTATES THE SEQUENCE OF AMINO ACIDS IN THE
FUNCTIONAL PROTEINS e.g. enzymes
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
AUG UAA
TRANSCRIPTION
single stranded mRNA
double stranded DNA
Functional Protein
TRANSLATIONprotein coding sequence
or open reading frame
MAPSSRGG…..
MOLECULAR BIOLOGY – DNA structure, genetic code
Mutations are variations in the DNA sequence e.g. single base pair substitution
ATG GCT CCT TCT TCC AGA GGT GGC . . . . . . TAATAC CGA GGA AGA AGG TCT CCA CCG . . . . . . ATT
5’5’3’
3’
AUG GCU CCU UCU UCC AGA GGU GGC . . . . . . UAA
SUCH MUTATIONS CAN INFLUENCE THE FUNCTIONALITY OF THE PROTEIN e.g. changing
which amino acid is incorporated
ATG GCT CCT TCA TCC AGA GGT GGC . . . . . . TAATAC CGA GGA AGT AGG TCT CCA CCG . . . . . . ATT
5’5’3’
3’
AUG GCU CCU UCA UCC AGA GGU GGC . . . . . . UAA
MAPSSSGG…..
Functional Protein
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
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
How is DNA organised in the cell?
Bacterial DNA ~ 1 mm long
~ 1 m
1000 x more than
…thanks to mobilesno more twisted telephone cords!
Most bacterial DNA exists in a covalently closed circular form
SUPERCOILING
MOLECULAR BIOLOGY – DNA structure, genetic code
protein scaffold
A typical bacterial chromosome consists of about 50 giantsupercoiled loops of DNA
TOPOISOMERASES – enzymes that insert or remove supercoils
Type I … break only one strand -> relaxing or twisting of the helixType II … break both strands and pass another part of the double helix through the gap
MOLECULAR BIOLOGY – DNA structure, genetic code
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
H2A, H2B, H3, H4
200 bp
80 bp
80 bp
40 bp
Nucleosome
MOLECULAR BIOLOGY – DNA structure, genetic code
Eukaryotic chromatin hierarcheal structure
Net result is that a eukaryotic (human) cell’s DNA is packaged into a mitotic chromosome 10,000 fold shorter than it extended length!
‘beads on a string’
30nm ‘solenoid fibre’
scaffold associated fibres
Condensed chromosome
SECOND SUMMARY – CRUCIAL KNOWLEDGE
MOLECULAR BIOLOGY – DNA structure, genetic code
T A C C G T T A G T T C A C G A T T
A T G G C A A T C A A G T G C . . . . . . T A ASTART STOP
CODING SEQUENCE
A U G G C A A U C A A G U G C U A A
RNA
densely packed chromosome
part of the chromosomewhere gene X is located
A U G G C A A U C A A G U G C U A ARNA
TRANSCRIPTION DNA RNA
MetAla
IleLys
AlaPROPERLY FOLDED PROTEINexecutes its function in cell
double-strand
DNA
MOLECULAR BIOLOGY – DNA structure, genetic code
The Central Dogma
TRANSLATIONRibosomes, tRNAs (expanded later)