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



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’


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


Watson moved to Cambridge in order to learn X-ray diffraction/

crystalography

X-ray crystalography


‘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


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


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


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


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!


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)


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)


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)


• 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


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


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


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!)


Nature April 25, 1953.

5-carbon sugar

Nitrogen containing base

Phosphate

4 bases: adenine, guanine, thymine, cytosine

DEOXYRIBONUCLEIC ACID - DNA


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


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









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


How do we experimentally test these theories?

- proposed by Watson and Crick

DNA replication: Meselson-Stahl experiment

normal 14N


• 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)


http://www.sumanasinc.com/webcontent/animations/content/meselson.html

Meselson-Stahl experiment video/ tutorial








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


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


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…..


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







AUG UAA

TRANSCRIPTION

single stranded mRNA

double stranded DNA

Functional Protein

TRANSLATIONprotein coding sequence

or open reading frame

MAPSSRGG…..


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


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


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


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



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


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


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


The Central Dogma

TRANSLATIONRibosomes, tRNAs (expanded later)

Documents

DNA STRUCTURE, GENETIC CODE, CHROMOSOMES