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Outline of Course: "Transmission of Genetic information:

1) Nucleic acids, composition and structure.

2) DNA replication.
3) Transcription.
4) Transcription in eucaryotes, splicing.
5) The genetic code.
6) Translation.
7) Molecular basis of mutations and DNA repair.
8) Control of gene expression; - the Operon.
9) Viruses; - Classes, RNA virus replication, Retroviruses.
10) Recombinant DNA technology.

Reading: Meisenberg (Recommended)

Additional: Harpers Biochemistry, 23rd, 24th, 25th Edition. Murray et al.,
Biochemistry, Stryer.

Other Reading: Lecture Handouts (Dr Kinsella)

MedGenetics Tutorial (Dr Kinsella)

Internet sites: Access Excellence: http://www.accessexcellence.org/

Graphics Gallery: http://www.accessexcellence.org/AB/GG/

Transmission of Genetic Information

Overview: Gene: -basic genetic unit - made of DNA

-codes for protein (or RNA) molecule
Genes -organised into Chromosomes.
All chromosomes of organism = Genome.
Replication
DNA DNA
Cell division
Transcription

[RNA]

Translation

Protein
Receptors Contractile Proteins
Hormones
Structural Proteins Enzymes
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1) Nucleic acids -composition and structure:

2 types of Nucleic Acid : DNA -Deoxyribonucleic acid

RNA - Ribonucleic Acid.

Basic components of DNA and RNA

Polymers - Nitrogen Base + 5 carbon Sugar + Phosphate

Bases -Purines = Adenine, Guanine in DNA and RNA

-Pyrimidines = Thymine, Cytosine in DNA;

Uracil, Cytosine in RNA.
6 7 4
5
1
3 5
8
2
4 2 1 6
3 9

Sugars - 5 carbon sugars : β- D- ribose in RNA

(Pentoses) β- D- 2-deoxyribose in DNA.

5
HOCH2
O OH
5
4 HOCH2
1
O OH
H H 4
1
3 2 H H
OH OH 3 2
OH H

(OH)in RNA
(H) in DNA
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Nucleoside - identified by its Base.

NH2
NH2
C 7
1 N 6 5 N C
C 4
3N 5 CH
2 C8
HC 2
C C 1 6C
N 4 9 N
O
3 N
HOCH2
HOCH2 O
O
H H
H H
OH OH (H)
OH OH (H)
CYTIDINE (DEOXYCYTIDINE)

ADENOSINE (DEOXYADENOSINE)

3 types of Nucleotides - No of Phosphates ??.

Nucleotide Monophosphate -NMP (dNMP) - 1 phosphate

Di -NDP (dNDP) -2
Tri -NTP (dNTP) -3

dATP
N1 Pyrimidine

N9 Purine

tri di mono
γ β α

Ribonucleotides - AMP, GMP, CMP, UMP & di/tri phosphate forms (No TMP)
Deoxyribonucleotides -dAMP, dGMP, dCMP, dTMP & di/tri phosphate forms (No dUMP)
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Oligonucleotide:
less than 100 nucleotides - joined by 3'-5' PD bonds.

Nucleic acid - Polynucleotide

-large numbers of nucleotides - joined by 3'-5' PD bonds.

Nucleic acid (DNA or RNA) has polarity:

-one end has a free 5'OH group
- the other has a free 3'OH group

5’
A
Orientation of the strands ???

3’-5’ PD
G
Identified by free OH groups
on the sugar;
3’-5’ PD
T

i.e the direction of one strand is

3’-5’ PD 5' - 3'.
C

3’

Sequence of nucleotides - given in 5'-3' direction unless otherwise

specified e.g sequence AGCAT = 5' AGCAT 3'
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2. -Bases are stacked on inside of helix;
-phosphate-deoxyribose units = backbone of α-helix.
-planes of the bases - perpendicular to the helix axis;
-planes of sugars - almost at right angles to those of the bases.

5’
3’

3’ 5’

4. Watson and Crick base pairing rules.

- 2 chains held by Hydrogen bonds between pairs of Bases on opposite strands.

-Steric restrictions of α-helical sugar-phosphate backbone determines base pairing rules:

•Adenine(A) pairs Thymine (T) -2 hydrogen bonds
•Guanine(G) pairs Cytosine (C) -3 hydrogen bonds
Guanine Cytosine
Adenine Thymine

Where rules correctly applied = Complementarity between the bases.

e.g. A is complementary to T,
C is complementary to G, and vice versa.
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DNA exists as a right handed α helix - (B-DNA)

- has two unequal Groves due to way: Bases stack &

Sugar – Phosphate backbone twists
20 Å
5’ 3’

3.4 Å Minor Groove

34Å
Major Groove

Sugar-phosphate backbone

Base pair

1 nm = 10 Å

3’ 5’

W & C base pairing rules - may also be applied to RNA.

In general, DNA is double stranded (d.s)
RNA is single stranded (s.s)

Exceptions e.g. -viruses can have d.s DNA, s.s DNA, d.sRNA, s.sRNA

Of the 3 types of RNA found in the cell, -all SS - but can form base pairs :
- e.g transfer RNA (tRNA) - has intramolecular base pairs

- 3D structure of a tRNA molecule.

Where base pairing occurs in RNA:

•A = Uracil -2 H bonds (T - not in RNA)

•G = Cytosine - 3 H bonds
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DNA replication:
- how the sequence of nucleotides (bases) in a genome is copied during cell division ???.

•Clues: W& Crick base pairing -Complementarity between bases

"if one were to write down the sequence of bases in one strand of DNA, one immediately
knows the sequence of bases of the strand to which it is base paired".

Mechanism: -one strand is a Template for the other during DNA replication.

DNA replication is Semi-Conservative - one strand is newly synthesized

- the other derived from Parent or template DNA.

Identical DNA
Sequence

Parental DNA Daughter DNA

Requirements of DNA Pol 1 to synthesize DNA:

1. 4 dNTPs; plus Mg2+

2. Growing strands of DNA must have a free 3' OH group.

Growing strand = PRIMER STRAND

DNA Pol.1 adds dNTPs onto 3'OH terminus of Primer Strand by catalyzing new 3'-5' PD + PPi

3. Incoming dNTP must be Complementary to the base on the Template strand.

-the template can be single or double stranded - s.s or d.s
- d.sDNA - effective only if sugar-phosphate backbone is broken at one or more sites.

5’ 5’ 5’
C C C 3’ G
G G 3’ G
T 3’ C C
T dCTP T dGTP T
A T T
T A A
A T Pol I T
A Pol I A A
T A A
C T T
G C C
G G G
C G G
C C
3’ 5’ 3’ 5’ 3’ 5’
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Mechanism??
Nucleophilic attack by the 3'OH terminus of Primer on α phosphate of incoming dNTP
3’
Primer
Strand

Primer
O Strand

CH2 Base O
O

H
CH2 Base
H
O
H
3’ OH.. H H
PPi
O O O H
3’ O
-
O–P– O–P– O–P– O
5’ - 3’ -O –P–O
Direction PD
- -
O -O O -
O
5’
CH2 Base 5’
CH2 Base
O
O

H H H H
3’ OH H 5’
OH H 5’
Template
Strand Template
Strand

DNA polymerase 1 elongation reaction proceeds in 5'-3' direction &

occurs only if incoming dNTP is complementary to base on the Template strand.

3'-5'exonuclease activity of DNA Pol. 1.

- if non-complementary base is incorporated, DNA Pol 1 recognizes it & corrects it.

3'-5'exonuclease - hydrolyse 3'-5' PD bond of DNA progressively from 3'OH terminus

- removes that nucleotide -as dNMP.

5’ 3’ 5’ 3’
– G - -C – – G - -C –
– A - -T – – A - -T –
– G - -C – – G - -C – Nucleotide removed must:
– T - -A – – T - -A –
- have a free 3'OH group
– C - -G – 3’ – 5’ – C - -G –
– A - -T – Exonuclease – A - -T – - be non-complementary to Template
– G - -C – – G - -C –
–C A– 3’HO
A–
3’ T– T–
C– C–
G– G–
5’ 5’