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Biochemistry, part 2

Biochemistry, part 2. Course outline. DNA folding. DNA folding.

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Biochemistry, part 2

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  1. Biochemistry, part 2

  2. Course outline

  3. DNA folding

  4. DNA folding • Many DNA molecules are circular (e.g., bacterial chromosomes, all plasmid DNA). Circular DNA can form supercoils. Human chromosome contains 3x109 basepairs and are wrapped around proteins to form nucleosomes. Nucleosomes are packed tightly to form helical filament, a structure called chromotin. • RNA are much shorter but more diverse molecules. They can form various three dimensional structures.

  5. Tertial structure in DNA • Supercoils refer to the DNA structure in which double-stranded circular DNA twists around each other. Supercoiled DNA contrasts relaxed DNA; • In DNA replication, the two strands of DNA have to be separated, which leads either to overwinding of surrounding regions of DNA or to supercoiling; • A specialized set of enzymes (gyrase, topoisomerases) is present to introduce supercoils that favor strand separation; • The degree of supercoils can be quantitatively described.

  6. Varieties of supercoiled DNA

  7. Linking number • The linking number L of DNA, a topological property, determines the degree of supercoiling; • The linking number defines the number of times a strand of DNA winds in the right-handed direction around the helix axis when the axis is constrained to lie in a plane; • If both strands are covalently intact, the linking number cannot change; • For instance, in a circular DNA of 5400 basepairs, the linking number is 5400/10=540, where 10 is the base-pair per turn for type B DNA.

  8. The twist and writhe • Twist T is a measure of the helical winding of the DNA strands around each other. Given that DNA prefers to form B-type helix, the preferred twist = number of basepair/10; • Writhe W is a measure of the coiling of the axis of the double helix. A right-handed coil is assigned a negative number (negative supercoiling) and a left-handed coil is assigned a positive number (positive supercoiling). • Topology theory tells us that the sum of T and W equals to linking number: L=T+W • For example, in the circular DNA of 5400 basepairs, the linking number is 5400/10=540 • If no supercoiling, then W=0, T=L=540; • If positive supercoiling, W=+20, T=L-W=520;

  9. The relation between L, T and W Positive supercoiling

  10. The relation between L, T and W Negative supercoiling

  11. L, T and W calculation • A relaxed circular, double stranded DNA (1600 bps) is in a solution where conditions favor 10 bps per turn. What are the L, T, and W? • During replication, part of this DNA unwinds (200 bps) while the rest of the DNA still favor 10 bps per turn. What are the new L, T, and W? 1400 bps 1600 bps 200 bps L=1600/10=160 W=0 (relaxed) T=L-W =160 L=160 T=(1600-200)/10=140 W=L-T=+20

  12. Nucleosomes • Nucleosomes look like “beads on a string” under microscope. The beads contain a pair of four histone proteins, H2A, H2B, H3, and H4 (octamer). The string is double stranded DNA; • The surface of the octamer contain features that guide the course of DNA such that DNA can wrap 1.65 turns around in a left-handed conformation. H1 proteins serves to seal the ends of the DNA and connects consecutive nucleosomes. nucleosomes

  13. Organisation of chromosomes Base pairs per turn Packing ratio 10 1 80 6-7 DNA double helix 2 nm ‘Beads on a string’ chromatin form 11 nm

  14. Organisation of chromosomes Base pairs per turn Packing ratio 1200 ~40 60,000 680 Solenoid (6 nucleosomes per turn) 30 nm Loops (50 turns per loop) o.25 μm

  15. Organisation of chromosomes Miniband (18 loops) Base pairs per turn Packing ratio 1.1 106 1.2 104 o.84 μm Chromosome (stacked minibands) o.84 μm

  16. Organisation of chromosomes

  17. Organisation of chromosomes

  18. proteins

  19. Genetic code • 4 possible bases (A, C, G, U) • 3 bases in the codon • 4 x 4 x 4 = 64 possible codon sequences • Start codon: AUG • Stop codons: UAA, UAG, UGA • 61 codons to code for amino acids (AUG as well) • 20 amino acids – redundancy in genetic code

  20. Amino acids • building blocks for proteins (20 different) • vary by side chain groups • Hydrophilic amino acids are water soluable • Hydrophobic are not • Linked via a single chemical bond (peptide bond) • Peptide: Short linear chain of amino acids (< 30) polypeptide: long chain of amino acids (which can be upwards of 4000 residues long).

  21. Glycine (G, GLY) Alanine (A, ALA) Valine (V, VAL) Leucine (L, LEU) Isoleucine (I, ILE) Phenylalanine (F, PHE) Proline (P, PRO) Serine (S, SER) Threonine (T, THR) Cysteine (C, CYS) Methionine (M, MET) Tryptophan (W, TRP) Tyrosine (T, TYR) Asparagine (N, ASN) Glutamine (Q, GLN) Aspartic acid (D, ASP) Glutamic Acid (E, GLU) Lysine (K, LYS) Arginine (R, ARG) Histidine (H, HIS) START: AUG STOP: UAA, UAG, UGA 20 amino acids

  22. 20 amino acids

  23. 20 amino acids

  24. The basic amino acid

  25. Peptide bond Two amino acids Removal of water molecule Peptide bond Formation of CO-NH Amino end Carboxyl end

  26. Peptide bond

  27. Polypeptide

  28. Protein structure There are four basic levels of structure in protein architecture

  29. Protein structure • Primary–sequence of amino acids constituting the polypeptide chain • Secondary–local organization into secondary structures such as  helices and  sheets • Tertiary –three dimensional arrangements of the amino acids as they react to one another due to the polarity and resulting interactions between their side chains • Quaternary–number and relative positions of the protein subunits

  30. Protein structure Primary structure: amino acid sequence

  31. Protein structure Secondary structure: α-helix and β-sheet Amino end Carboxyl end

  32. Protein structure Secondary structure: α-helix and β-sheet Parallel Antiparallel Side view Side view

  33. Protein structure Secondary structure: α-helix and β-sheet

  34. Protein structure Tertiary structure: spatial arrangement of amino residues

  35. Protein structure Quaternary structure: spatial arrangement of subunits

  36. Protein structure primary secondary tertiary quaternary

  37. Protein structure

  38. Protein function • Every function in the living cell depends on proteins. • Motion and locomotion of cells and organisms depends on contractile proteins. [Example: Muscles] • The catalysis of all biochemical reactions is done by enzymes, which contain protein. • The structure of cells, and the extracellular matrix in which they are embedded, is largely made of protein. [Example: Collagens] • Defence by antibodies. • The receptors for hormones and other signalling molecules are proteins. • The transcription factors that turn genes on and off to guide the differentiation of the cell and its later responsiveness to signals reaching it are proteins. • and many more - proteins are truly the physical basis of life.

  39. Protein function

  40. Protein function antibody

  41. Protein function enzyme

  42. Gene expression

  43. Gene regulation mechanism Bacteria express only a subset of their genes at any given time. • Expression of all genes constitutively in bacteria would be energetically inefficient. • The genes that are expressed are essential for dealing with the current environmental conditions, such as the type of available food source.

  44. Gene regulation mechanism Regulation of gene expression can occur at several levels: • Transcriptional regulation: no mRNA is made. • Translational regulation: control of whether or how fast an mRNA is translated. • Post-translational regulation: a protein is made in an inactive form and later is activated.

  45. Gene regulation mechanism Post-translational control Transcriptional control Translational control Lifespan of mRNA Protein activation (by chemical modification) Protein Translation rate Onset of transcription Feedback inhibition (protein inhibits transcription of its own gene) Ribosome mRNA DNA RNA polymerase

  46. Escherichia.Coli

  47. Gene regulation mechanism Operon • A controllable unit of transcription consisting of a number of structural genes transcribed together. Contains at least two distinct regions: the operator and the promoter.

  48. Gene regulation mechanism Case study of the regulation of the lactose operon in E. coli • E. coli utilizes glucose if it is available, but can metabolize other sugars if glucose is absent.

  49. Gene regulation mechanism Food source: Glucose : Lactose Glucose : Lactose Glucose : Lactose 1:3 1:1 3:1 Second period of rapid growth with lactose as food source 70 60 29.5 50 14.0 40 43.5 Relative density of cells 30 20 26.5 Initial period of rapid growth with glucose as food source 39.0 13.5 10 0 0 2 3 4 5 0 1 3 4 5 6 0 1 2 1 2 3 4 5 6 7 Time (hours)

  50. Gene regulation mechanism Case study of the regulation of the lactose operon in E. coli • Genes that encode enzymes needed to break other sugars down are negatively regulated. • Example: enzymes required to metabolize lactose are only synthesized if glucose is depleted and lactose is available. • In the absence of lactose, transcription of the genes that encode these enzymes is repressed. How does this occur?

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