1. TOPICS IN (NANO) BIOTECHNOLOGY Lecture II 3 march 2004 PhD Course

2. 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?

3. 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

4. Brief history of genetics

5. The central dogma Protein, a linear sequence of amino acids is encoded by DNA, a linear sequence of nucleotides

6. The central dogma

7. Where to begin? Nucleic Acids Information Storage, Retrieval & Use Information Content nitrogenous bases purines pyrimidines Scaffolding ribose+phosphate Nucleotides Repeat Units

8. 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

9. 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

10. 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 2-deoxy-D-ribose (in DNA) D-ribose (in RNA) 1’ 2’ 3’ 4’ 5’

11. 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 1 2 3 4 5 6

12. Cytosine 2-oxy-4-amino pyrimidine Used in both DNA & RNA Thymine 2,4-dioxy-5-methyl pyrimidine (DNA only) Uracil 2,4-dioxy pyrimidine (RNA only) (Root underlined) The pyrimidine bases

13. 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 N H N 1 2 4 5 6 7 8 9 3 The purine bases

14. Found in both DNA and RNA (Root underlined) 6-aminopurine2-amino-6-oxy purine The purine bases

15. 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  -N 1 -glycosidic bonds in pyrimidine ribonucleosides  -N 9 -glycosidic bonds in purine ribonucleosides

16. Nucleotides= nucleoside + phosphate Nucleotides are polyprotic acids (eg Adenosine 5’-monophosphate AMP)

17. Functions of Nucleotides Precursors to the polynucleotides DNA & RNA Carriers of energy via phosphoryl group transfer e.g. ATP + H 2 O  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

18. 3’ 5’ Linking Nucleotides by 5’-3’ Phosphodiester Bonds

19. +H 2 O Linking Nucleotides by 5’-3’ Phosphodiester Bonds

20. 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

21. DNA structure & function DNA 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

22. DNA double helix Base-pairing – spontaneous process called hybridisation Thermodynamically controlled > T m - melting Anti-parallel, by convention 5’-3’ always specified

23. Denaturation & renaturation Denatured by heating to above melting temperature – T m is defined as the midpoint of the temperature range for denaturation By cooling – renaturation or annealing can occur

24. 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 Absorbance

25. DNA structure & function

26. 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

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

29. DNA strands separate at the replication fork Replication