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Biochemistry, part 2 Course outline 1 Introduction 2 Theoretical background Biochemistry/molecular biology 3 Theoretical background computer science 4 History of the field 5 Splicing systems 6 P systems 7 Hairpins 8 Micro technology introductions Microreactors / Chips 9 Microchips and fluidics 10 Self assembly 11 Regulatory networks 12 Molecular motors 13 DNA nanowires 14 Protein computers 15 DNA computing - summery DNA folding 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. 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 quantitatively described. can be Varieties of supercoiled DNA 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 righthanded 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. 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 lefthanded 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; The relation between L, T and W Positive supercoiling The relation between L, T and W Negative supercoiling 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? 1600 bps L=1600/10=160 W=0 (relaxed) T=L-W =160 1400 bps 200 bps L=160 T=(1600-200)/10=140 W=L-T=+20 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 nucleosomes. DNA and connects consecutive nucleosomes Organisation of chromosomes Base pairs per turn DNA double helix Packing ratio 2 nm 10 1 11 nm 80 6-7 ‘Beads on a string’ chromatin form Organisation of chromosomes Solenoid (6 nucleosomes per turn) Base pairs per turn Packing ratio 30 nm 1200 ~40 60,000 680 Loops (50 turns per loop) o.25 μm Organisation of chromosomes Miniband (18 loops) Base pairs per turn o.84 μm Chromosome (stacked minibands) o.84 μm 1.1 106 Packing ratio 1.2 104 Organisation of chromosomes Organisation of chromosomes proteins 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 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). 20 amino acids 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 20 amino acids The basic amino acid Peptide bond Two amino acids Removal of water molecule Peptide bond Formation of CO-NH Amino end Carboxyl end Peptide bond Polypeptide Protein structure There are four basic levels of structure in protein architecture 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 Protein structure Primary structure: amino acid sequence Protein structure Secondary structure: α-helix and β-sheet Amino end Carboxyl end Protein structure Secondary structure: α-helix and β-sheet Parallel Antiparallel Side view Side view Protein structure Secondary structure: α-helix and β-sheet Protein structure Tertiary structure: spatial arrangement of amino residues Protein structure Quaternary structure: spatial arrangement of subunits Protein structure primary secondary tertiary quaternary Protein structure 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 signals reaching it are proteins. and many more - proteins are truly the physical basis of life. to Protein function Protein function antibody Protein function enzyme Gene expression 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. Gene regulation mechanism Regulation of gene several levels: expression can occur at 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. Gene regulation mechanism Transcriptional control Translational control Post-translational control Lifespan of mRNA Protein Onset of transcription Translation rate Ribosome mRNA DNA RNA polymerase Protein activation (by chemical modification) Feedback inhibition (protein inhibits transcription of its own gene) Escherichia .Coli 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. Gene regulation mechanism Case study of the operon in E. coli regulation of the lactose E. coli utilizes glucose if it is available, but can metabolize other sugars if glucose is absent. Gene regulation mechanism Food source: Glucose : Lactose Glucose : Lactose 1:3 Glucose : Lactose 1:1 70 60 50 40 30 20 3:1 29.5 14.0 43.5 26.5 39.0 13.5 10 0 0 1 2 3 4 5 0 1 2 3 4 5 Time (hours) 6 0 1 2 3 4 5 6 7 Second period of rapid growth with lactose as food source Initial period of rapid growth with glucose as food source Gene regulation mechanism Case study of the operon in E. coli regulation of the lactose 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? Gene regulation mechanism Case study of the regulation of the lactose operon in E. coli All the loci required for lactose metabolism are grouped together into an operon. The lacZ locus encodes -galactosidase enzyme, which breaks down lactose. The lacY locus encodes galactosidase permease, a transport protein for lactose. The function of the lacA locus is unknown. The lacI locus encodes a repressor blocks transcription of the lac operon. that Gene regulation mechanism Regulatory function Cleaves lactose to glucose and galactose Regulatory protein Lacl ß-galactosidase LacZ Membrane transport protein-imports lactose Galactosidase permease LacY Section of E. coli chromosome lacl lacZ Observations about regulation of lacZ and lacY: (1) Lacl protein and glucose shut down transcription of lacZ and lacY Glucose Lactose E. coli Galactose (2) Lactose induces transcription of lacZ andlacY lacY Galactosidase permease Chromosome ß-galactosidase Gene regulation Lac operon Lac operon lacl promoter lacl Promoter Operator lacZ lacY lacA Gene regulation mechanism Repression and induction of the lactose operon. The lac operon is under negative regulation, i.e. , normally, transcription is repressed. Glucose represses transcription of the lac operon. Glucose inhibits cAMP synthesis in the cells. At low cAMP levels, no cAMP is available to bind CAP. Unless CAP is bound to the CAP site in the promoter, no transcription occurs. Gene regulation mechanism When no lactose is present, the repressor binds to DNA and blocks transcription. NO TRANSCRIPTION Functional repressor lacl lacZ RNA polymerase blocked Operator (binding site for repressor) lacY Gene regulation mechanism Repressor plus lactose (an inducer) present. Transcription proceeds. Lactose TRANSCRIPTION BEGINS repressor lacl + mRNA Permease galactosidase lacZ lacY Gene regulation mechanism Operons produce mRNAs that code for functionally related proteins. "Polycistronic" mRNA lacZ message RNA polymerase binds to promoter lacY message lacA message lacl promoter lacl Promoter Operator lacZ lacY lacA DNA binding sites DNA binding proteins Proteins that bind to DNA share similarity in the structure of their DNA-binding regions. Many DNA binding proteins, such as lac repressor, have a helix-turn-helix motif which fits into the major groove of a DNA molecule DNA binding proteins (a) (b) (c) DNA binding proteins Binding of an inducer to the lac repressor causes it to release the operator DNA because it alters the conformation of the helix-turn-helix motif. DNA binding proteins DNA binding proteins DNA binding proteins DNA binding proteins Information about regulation of the expression of genetic loci may help to combat diseases. Virulent bacterial strains have genes that encode the ability to infect and produce disease. Knowledge of how the expression of these genes is controlled and regulated may provide insights into blocking the development of the disease. DNA binding proteins, negative regulation When tryptophan is absent, transcription occurs. RNA polymerase Leader 5 coding loci Promoter When tryptophan is present, transcription is blocked. Tryptophan Repressor DNA binding proteins Ribosomes translates mRNA rapidly when tryptophan is abundant,… …leading to formation of stem-and-loop structure that inhibits RNA polymerase and terminates transcription.