TOPICS IN (NANO) BIOTECHNOLOGY

Lecture II

3 march 2004

PhD Course

Overview• Definitions • What is DNA? What are nucleic acids? • What are nucleotides and nucleosides?• What are chromosomes?• What is DNA replication, annealing and hybridisation?• What is translation and transcription?• What is the genetic code?• What are mutations and what are their effect?

Definitions• Genome – complete set of sequences in the genetic material of an organism

• Nucleic acids are molecules that encode genetic information

• A gene is the segment of DNA involved in producing a polypeptide chain • A locus is the position on a chromosome at which the gene for a particular trait resides

• An allele is one of the several alternative forms of a gene occupying a locus

Brief history of genetics

The central dogma

Protein, a linear sequence of amino acidsis encoded by DNA, a linear sequence of nucleotides

The central dogma

Where to begin?

Nucleic AcidsInformation Storage, Retrieval & Use

Information Contentnitrogenous bases

purinespyrimidines

Scaffoldingribose+phosphate

NucleotidesRepeat Units

What are nucleic acids? • DNA was first isolated from the nuclei; it is an acid

because of its phosphate groups – hence the term nucleic acid.

• It contains a sugar, 2-deoxy-D-ribose and thus is called deoxyribonucleic acid, DNA

• There is another acid of similar structure that is found in cells in which the sugar is D-ribose and is thus called ribonucleic acid, RNA

• DNA contains the genetic information of most organisms and RNA is involved in the expression of the information contained in DNA

Nucleotides• The building block of a nucleic acid is a nucleotide.

•Nucleotide – nucleoside linked to a phosphate group at either the 3’ or 5’ position of the pentose sugar

•Nucleoside - purine or pyrimidine base linked to position 1 of a pentose sugar

• DNA contains the four bases adenine, guanine, cytosine and thymine; RNA has uracil instead of thymine

Pentoses of Nucleotides

Riboses are one component of the scaffolding for nucleic acids

The difference: 2'-OH vs 2'-H

This difference influences– the secondary structure of RNA & DNA– the stability of RNA and DNA

O

H

OH

H

OH

H

OH

CH2

H

OHO

H

OH

H

H

H

OH

CH2

H

OH

2-deoxy-D-ribose (in DNA)D-ribose (in RNA)

1’ 1’

2’ 2’3’ 3’

4’4’

5’5’

• All are built on the pyrimidine platform

• Ring is numbered to assign the lowest possible numbers to the two nitrogens

• Connection to the ribose sugar is via a glycosidic bond from position 1

• All have an oxygen bonded to position 2(i.e. all are 2-oxo- substituted pyrimidines)

• Position 4 will bear an oxo or amino group

• Position 5 is methyl-substituted in one case

The pyrimidine bases

N

N

1

2

3

4

5

6

R

N

N

NH2

O

R

N

NH

O

O

CH3

R

N

NH

O

O

Cytosine2-oxy-4-amino pyrimidineUsed in both DNA & RNA

Thymine2,4-dioxy-5-methyl pyrimidine

(DNA only)

Uracil2,4-dioxy pyrimidine(RNA only)

(Root underlined)

The pyrimidine bases

• All are built on the purine platform

• Ring is numbered to assign the lowest possible numbers to the four nitrogens

• Connection to the ribose sugar is via a glycosidic bond from position 9

• 6-membered ring will be oxo or amino-substituted at positions 2 or 6

N

N

NH

N1

24

56 7

8

93

The purine bases

Adenine R

N

N

N

N

NH2

Guanine R

N

NH

N

N

NH2

O

Found in both DNA and RNA (Root underlined)

6-aminopurine 2-amino-6-oxy purine

The purine bases

NO

OH

OHOH

N

NN

NH2

Nucleosides = base+pentose• Base is linked via a glycosidic bond

• Named by adding: -idine to the root name of a pyrimidine -osine to the root name of a purine

• Sugars make nucleosides more water-

soluble than the free bases they bear

• -N1-glycosidic bonds in pyrimidine ribonucleosides

• -N9-glycosidic bonds in purine ribonucleosides

N

OOH

OHOH

NH

O

O

N

OO

OHOH

N

NN

NH2

P

O

OH

OH

Nucleotides= nucleoside + phosphate

Nucleotides are polyprotic acids

(eg Adenosine 5’-monophosphate AMP)

Functions of Nucleotides• Precursors to the polynucleotides DNA & RNA

• Carriers of energy via phosphoryl group transfer

e.g. ATP + H2O ADP + Pi + energy

– bases serve as recognition units

• ATP is central to energy metabolism • GTP drives protein synthesis • CTP drives lipid synthesis • UTP drives carbohydrate metabolism

• Cyclic nucleotides are signal molecules and regulators of cellular metabolism and reproduction

N

OO

OH

OH

OH OP

N

NN

NH2

3’

N

OO

OH

OH

OH OP

N

NHN

NH2

O

5’

Linking Nucleotides by 5’-3’ Phosphodiester Bonds

N

OO

O

OH

OH OP

N

NN

NH2

N

OO

OH

OH OP

N

NHN

NH2

O

+H2O

Linking Nucleotides by 5’-3’ Phosphodiester Bonds

N

OO

O

O-

O-

OP

N

OO

O

O-

OP

N

OO

O

O-

OP

N

OO

OH

O-

OP

N

NHN

NH2

O

N

NN

NH2

N

NH2

O

N

NN

NH2

The sample shown here is a DNA molecule with the sequence 5’-GACA-3’. The arrow gives the direction of the chain

5’

3’

• Polymers linked 5’ to 3’ by phosphodiester bridges

• Sequence is always read 5' to 3' • In terms of genetic information, this

corresponds to "N to C" in proteins• phosphodiester is weakly acidic:

dissociated at neutral pH anionic

Nucleic acids: linear polymers of nucleotides

DNA structure & functionDNA is a double helix

- one type, one purpose: genetic material

- Base pairing is the specific interaction of adenine with thymine or guanine with cytosine

- Complementary base pairs are A-T, G-C (or in the case of RNA, A-U)

- Anti-parallel strands of the double helix are organised in opposite orientation, so that the 5’ end of one strand is aligned with the 3’ end of the other strand

DNA double helix

Base-pairing – spontaneous process called hybridisation

Thermodynamically controlled > Tm - melting

Anti-parallel, by convention 5’-3’ always specified

Denaturation & renaturation

Denatured by heating to above melting temperature – Tm is defined as the midpoint of the temperature range for denaturation

By cooling – renaturation or annealing can occur

Bases absorb in the UV-region• Aromatic, max~ 260 nm

• useful for:– quantifying nucleic acids– assessing purity– monitoring structural changes (eg melting of double-stranded

DNA)

220 260 300

Wavelength, nm

Abs

orba

nce

DNA structure & function

RNA

3 types, 3 purposes

Primary, secondary & tertiary structures all occur

• ribosomal RNA - the basis of structure and function of ribosomes

• messenger RNA - carries the message

• transfer RNA - carries the amino acids

• RNA is a single stranded molecule but can form duplexes with complementary RNA and DNA strand

• RNA is polarized and its synthesis proceeds in the 5’ to 3’ direction

RNA structure & function

Replication is the reproduction of genetic material and is semi-conservative

Replication

Replication

DNA strands separate at the replication fork

Replication

Replication