Questions from the last lecture. PHAR2811 Dale s lecture 3. Folate conversions. Prokaryotes. Prokaryotes

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1 PAR2811 Dale s lecture 3 Genome Structure MMWEALT F AUSTRALIA opyright Regulation WARIG This material has been reproduced and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of the opyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice Questions from the last lecture The role of methotrexate in the inhibition of thymidylate synthase Methotrexate is a competitive inhibitor of the enzyme that converts dihydrofolate (DF) to tetrahydrofolate (TF) (dihydrofolate reductase, DFR) It binds to the enzyme with ~1000 times the affinity of DF...making the inhibition almost irreversible Dihydrofolate (DF) Folate conversions DF reductase Tetrahydrofolate, (TF) TMP 2 2 R etc ADP + + Dihydrofolate, DF DF reductase Thymidylate ADP+ Synthase Thymidylate y synthase Serine hydroxymethyl transferase dump 2 Serine ydroxmethyl Transferase 2 5, 10 -methylene TF 2 Tetrahydrofolate, TF R etc 2 Serine 2 R Glycine etc This methylene group is transferred to dump at 6 Prokaryotes The genome of prokaryotes is extremely efficient. There are 4.6 million base pairs in your average E. coli If the average bacterial protein has a molecular weight of ~40,000 D how many different proteins does the average E. coli make? Prokaryotes To do this calculation you need to know: The average mol. Wt. of an amino acid ~100 This means the average protein has 400 amino acids Which means 1200 bases + promoter and terminator sequences ~1500 bp. 4.6 X 10 6 /1500 = ~3000 different proteins. 1

2 In humans if the whole genome was coding The genome has 3*10 9 bp and the average protein subunit is 50,000 So 500 aa = 1500 bp = 3000 bp with large promoter regions which makes the maths easy 3*10 ^9/3*10^3 = 1*10^6 or 1 million different proteins. We only make about 30,000 different proteins so there is a discrepancy Prokaryotes versus Eukaryotes Prokaryotes have no room for redundant sequences. Their survival depends on rapid proliferation when nutrients are available omplex multi-cellular eukaryotes depend for survival on quick responses, adjusting to changes in the environment. Prokaryotes versus Eukaryotes E. coli can divide every 20 min if conditions are optimal The human cell takes 18 to 24 h to go through the cell cycle once. The human genome only has about 2% coding regions. The gene density is much lower!! hromosome haracteristics hromosomes vary in number between species. The chromosome number is a combination of the haploid number (n) X the number of sets. Algae and fungi are haploid; most animals and plants are diploid. The number of pairs of chromosomes in different species genomes is bizarre. What do these life forms have in common? hromosome haracteristics Species Genome size in Megabases (Mb) # aploid chromosomes E. coli circular yeast cow 39 human alligator 16 carp 52 salamander

3 hromosome haracteristics hromosomes vary in size within a species. Within the human genome there is a four fold difference in the size of the chromosomes. entromere: the region of the chromosome where the spindle fibres attach. Repetitive satellite DA is often found around the centromere. Telomere: ends of the chromosome, containing a distinct repeating sequence, which enables the ends of the chromosome to replicate. entromere haracteristics The relative position of the centromere is constant, which means that the ratio of the lengths of the two arms is constant for each chromosome. This ratio is an important parameter for chromosome identification, and also, the ratio of lengths of the two arms allows classification of chromosomes into several basic morphologic types: entromere haracteristics hromosome Banding hromosomes can be stained with special dyes which give a consistent and unique pattern like a bar-code for each chromosome; so much so that the bands have been numbered. The most common stain used is a Giesma stain. This stain, when applied after mild proteolytic treatment (trypsin) gives light (G-light) and dark (G-dark) bands. The uman Genome 3

4 The ps and qs of chromosomes There are 2 arms on the chromosome denoted p and q For most chromosomes the short arm is the petit or p arm The longer arm is the q or queue arm umbering is done from the centromere along one of the arms hromosome Banding When viewed at the lowest resolution only a few bands appear. These are numbered p1, p2, p3 etc counting from the centromere. If the stained chromosomes are viewed at higher resolution many sub-bands bands are revealed. So the labelling then goes p11, p12, p13. So if your DA marker may be given a position on the chromosome with a set of numbers like 17p23. This means the locus is on chromosome 17 on the short p arm in sub-band 23. Some terms: The general material which makes up the chromosomes is called chromatin This is composed of DA and protein. eterochromatin ti contains DA which is more tightly packaged or condensed and probably is transcriptionally inert. Euchromatin contains most active genes; those actively transcribed. hromosome packaging at the molecular level. Each chromosome contains a single molecule of DA This DA is wound around small proteins called histones These proteins have lots of lysine and arginine residues, making them very positively charged at p 7 (and high pis ~12) istones istone ctamer There are 5 major histone variants: 1, 2A, 2B, 3 and 4. Two molecules each of 2A + 2B make up an octamer which the DA wraps around with 1.7 turns. This structure is known as a nucleosome. Each nucleosome has an 1 associated and a linker section of DA, like beads on a thread. 4

5 istones igher order Packaging The major force holding the association of histones to DA is electrostatic. To separate the histones from the DA, chromatin is treated with high ionic strength solutions. The high salt reduces the electrostatic interactions and the protein dissociates from the DA. Figure A model for chromosome structure, human chromosome 4. The 2-nm DA helix is wound twice around histone octamers to form 10-nm nucleosomes, each of which contains 160 bp (80 per turn). These nucleosomes are then wound in solenoid fashion with six nucleosomes per turn to form a 30- nm filament. In this model, the 30-nm filament forms long DA loops, each containing about 60,000 bp, which are attached at their base to the nuclear matrix. Eighteen of these loops are then wound radially around the circumference of a single turn to form a miniband unit of a chromosome. Approximately 10 6 of these minibands occur in each chromatid of human chromosome 4 at mitosis. The cell cycle At interphase (G1, S and G2) the chromosomes look like a plate of spaghetti, entangled and dispersed throughout the nucleus hromosomes at interphase At M phase the newly replicated daughter chromatids condense and line up. 5

6 The role of histones Shield the negative charges of the phosphates Allow bending and DA wrapping Restrict t access to transcription ti The interaction between the histones and the DA is dynamic and non-base sequence specific istone Remodeling Influences the DA accessibility for transcription an be one of the first events when switching on a set of genes. ucleosome remodeling complexes ucleosome positioning istone modification istone modifications Phosphorylation; serine residues Methylation, adding 3 Acetylation, lysine residues istone modifications Phosphorylation; serine residues Methylation, adding 3 Acetylation, lysine residues Acetylation Lysine Transferring an acetyl group to the amino side chain of lysine residues istone acetyl Transferases (ATs) istone deacetylases (DAs)

7 Acetylated Lysine What effect would acetylation have on DA accessibility? It neutralises the positive charge of the lysine side chain The histone will not have as much affinity i for the DA phosphates (negative) The nucleosome packing will be looser DA more accessible for transcription Deacetylases will pack it up again! 7