TOPICS IN (NANO) BIOTECHNOLOGY Lecture II

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

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

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

11. 4 3 5 6 2 1 The pyrimidine bases • 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

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

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

14. The purine bases 6-aminopurine 2-amino-6-oxy purine Found in both DNA and RNA (Root underlined)

15. Nucleosides = base+pentose • Base is linked via aglycosidic bond • Named by adding: -idineto the root name of apyrimidine -osineto the root name of apurine • Sugars make nucleosides more water-soluble than the free bases they bear • b-N1-glycosidic bondsin pyrimidine ribonucleosides • b-N9-glycosidic bondsin purine ribonucleosides

16. Nucleotides= nucleoside + phosphate Nucleotides are polyprotic acids (egAdenosine5’-monophosphateAMP)

17. 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

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

19. +H2O Linking Nucleotides by 5’-3’ Phosphodiester Bonds

20. Nucleic acids: linear polymers of nucleotides 5’ • 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 The sample shown here is a DNA molecule with the sequence 5’-GACA-3’. The arrow gives the direction of the chain 3’

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 > Tm - melting Anti-parallel, by convention 5’-3’ always specified

23. 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

24. Bases absorb in the UV-region • Aromatic, lmax~ 260 nm • useful for: • quantifying nucleic acids • assessing purity • monitoring structural changes (eg melting of double-stranded DNA) Absorbance 220 260 300 Wavelength, nm

25. DNA structure & function

26. RNA 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

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

28. Replication

29. Replication DNA strands separate at the replication fork

30. Replication