DNA Architecture and Physical Properties; Organellar DNA

1. DNA Architecture and Physical Properties; Organellar DNA AHMP 5406

2. Objectives: • Describe the molecular structure of DNA • Describe the packing and organization of DNA into eukaryotic chromosomes • Discuss the function, structure and components of nucleosomes • Understand the difference between nuclear and organellar codon usage

3. DNA Double Helical Structure

5. Eukaryotic Chromosomes • Consist of highly coiled DNA, RNA, histones and non-histone proteins • Chromosomes are numbered from largest to smallest 1 through 22, plus X and Y

6. Eukaryotic Chromosomes • Chromosomes are distinguished by: • size • centromere location • differential staining • DNA probes

7. Eukaryotic chromosomes • In metaphase of mitosis, chromosomes can be seen under microscope • they have a compact rod-like structure • The ends of chromosome are called telomeres, function is to protect the ends of the DNA

8. Eukaryotic chromosomes • Near the middle is the centromere • Function is to attach to spindles during cell division • Ensure correct segregation • Telomeres and centromeres contain special DNA sequences and associated proteins

9. Eukaryotic chromosomes • Telomeres are replicated differently from the rest of the genome • Different regions of the chromosome can be stained with dyes (e.g. Giemsa) giving a characteristic banding pattern

10. Eukaryotic chromosome structure (heterochromatin) Genes, repeated sequences, replication origins (mostly euchromatin) Telomeres (heterochromatin)

12. Chromatin packing ?

13. Histones – What are they??? • DNA packing material • Can be acetylated and interact in gene transcription • Methylation of lysines can inhibit or enhance transcription

14. Histones – What are they??? • Positively charged proteins – lots of Arg and Lys • Octameric • 5 different kinds – 4 are found in nucleosomes, one kind joins linker regions of DNA

15. Histones – What are they??? • Small proteins • 102-135 AA • Similar structural motif • Three a-helices • Connected by two loops • Histone-fold • H3-H4 dimer • H2A-H2B dimer

16. The nucleosome • DNA wrapped in 1.6 left-handed turns • DNA 146 bp • 11 nm in width

17. Bending of DNA around nucleosome • A-T rich sequences are easier to bend • Explains precise positioning of nucleosomes along DNA • Proteins also affect binding

18. Euchromatin vs. Heterochromatin

19. Heterochromatin • Is found in parts of the chromosome where there are few or no genes, such as • centromeres • telomeres • transposons and other "junk" DNA

20. Heterochromatin • Is densely-packed • Has reduced crossing over in meiosis • Those genes present in heterochromatin are generally inactive; that is, not transcribed…How?

21. Heterochromatin • Decreased acetylation of histones • Decreased methylationof lysine-9 in histone H3, • which now provides a binding site for heterochromatin protein 1 (HP1), • blocks access to transcription factors needed for gene transcription

22. Euchromatin • Found in parts of the chromosome that contain many genes • Loosely-packed in loops of 30-nm fibers

23. Euchromatin • Loops are separated from adjacent heterochromatin by insulators • Boundary sequences • Loops are often found near the nuclear pore complexes • Makes sense for the gene transcripts to get to the cytosol

24. Euchromatin • The genes in euchromatin are active and thus show • decreased methylation of the cytosines in CpG islands of the DNA • increased acetylation of histones and • decreased methylation of lysine-9 in histone H3.

25. Signaling by Modification of Nucleosome Histone Proteins

26. Modification of Histone tails • Histone tails are subject to covalent modification • Lysine • methylation (M) • acetylation (Ac) • Serine • phosphorylated (P) • Ubiquitin (U) • 76 AA protein

27. Modification of Histone tails • Histone acetyl transferases (HATs) • Add acetyl groups • Histone deacetylases (HDACs) • Remove acetyl groups • Affects stability of 30 nm fiber • Can attract specific proteins to modified chromatin • Some positions can be modified more than one way

28. Modification of Histone tails • Convey messages to the cell • E.g Chromatin is newly replicated • Or gene expression should not take place • Different modifications can attract different protein complexes • Allows chromatin to be dynamic

29. Modification of Histone tails • Only a few effects of specific modifications known

30. Examples of Histone tail mods • Gene expression • Ac of lysine (K) 14 on H3 • Ac of K 8 and 16 on H4 • P (S 10) and Ac (K 14) on H3

31. Examples of Histone tail mods • Gene silencing • Unmodified H3 and H4 • M of K 9 on H3

32. Other parts of the chromosome in detail

33. Centromeres • Essential for correct segregation of chromosomes • During cell division • By attachment to spindles • Consists of a small, core DNA sequence (AT rich) and specific proteins • a satellite DNA • In many species (e.g. humans) flanked by 100s of copies of a tandemly-repeated sequences

34. Centromeres • Packaged as heterochromatin • Has specialized packaging structures • Centromere-specific proteins • Centromere-specific nucleosomes • H3 variant = CENP-A • Facilitate construction of other centromere binding proteins • These attach to kinetochore

35. Telomeres • Repetitive sequence at the ends of chromosomes • TTAGGG • Many thousands of repeats • Lose a few repeat units with each replication cycle • Protects the genes contained in the chromosomes

36. The problem of telomere replication

37. More on genome organization

38. Compared to prokaryotes • Eukaryotic genomes are completely different in their organization and much larger • Their genes are mostly “split” into exons and introns • Exons may allow evolution of proteins in a “modular” way

39. Eukaryotic gene organization 5’UTR Exon1 Intron 1 Exon2 Intron 2 Exon 3 3’UTR Promoter Primary RNA transcript splicing mRNA Protein

40. The human genome is complex:

41. Repetitive/Mobile Elements

42. Origins and function of DNA classes • Highly repetitive: • Bits of old virus genomes • Simple sequence repeats e.g. CACACA…. • Special sequences such as centromeres • Moderately repetitive: • Other old virus genomes • Multi-gene families, e.g. ribosomal RNA • Single-copy: • Most “normal” genes

43. Types of repeated DNA • Tandemly repeated • Satellite DNA • Centromeric DNA • Minisatellites (10-100 bp) • E.g. (CAGACAGTATGA)n • DNA finger printing • Microsatellites (2-4 bp) • Can be used to study microevolution • Biparentally inherited • High rates of mutation • Alleles = size differences (CA)6 CA CA CA CA CA CA

44. Types of repeated DNA • Interspersed • SINES • Short interspersed nuclear elements • Approx. 500bp • Reversed transcribed RNA sequences • Non-coding • Ex. In humans Alu repeats (CA)6 CA CA CA CA CA CA

45. Types of repeated DNA • Interspersed • LINES • Long int. nucl. elem. • Approx. 5Kb • Code for two proteins • Reverse transcriptase and endonuclease activity • RNA binding ability • Increase genome size by copying themselves (CA)6 CA CA CA CA CA CA

46. Transposons • Move by “cut-n-paste” • Transposon is cut out of its location by an enzyme • Transposase is encoded within the transposon • Then inserted into a new location

47. Transposons and disease • Transposons are mutagens • Functional gene insertion • Can result in damage • Alter gene function • Inactivate protein • Faulty repair of the gap left at the old site • The string of identical repeats can cause problem in base pairing during mitosis • unequal crossing over

48. THE GENETIC CODE

49. The Genetic Code • Rules by which DNA encode AAs • Each codon = an AA • Thus determine polypeptide sequence • Protein function

50. The mitochondrial genome • Study of human mtDNA • Different code found • Other variants have been found • Bacteria