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Chap_24_2010 - Chemical Aspects of Biological Systems...

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Unformatted text preview: Chemical Aspects of Biological Systems Information Pathways (142C/242C) Meetin g Time: Ins tru ctor: TR, 12: 30- 1:4 5 P M, Phelps H all 3 515 Prof. L u c J aeger Co ntact: jaeg [email protected] chem. ucsb.edu Office: P S BN 464 9A Office Hrs: R , 2 :00 -3:00 PM; by ap pointm ent wade (wg [email protected] chem.ucs b.e du) PSBN 46 38, P hone 5 302 TR (W ad e) : 1 1:00 AM-12:00 AM an d by a ppointment TA: TA Office Hours : http://www.chem.ucsb.edu/coursepages/index.shtml Username: chem_142c and Password: chem_242c Informational pathways and metabolism: the example of the synthesis of the building blocks of proteins, RNA and DNA (Chap. 22 and 18) Information pathways and DNA (Chap. 24 and 25) Information pathways and RNA (Chap. 26) Information pathways and Proteins (Chap. 27) Information pathways and gene regulation (Chap. 28) Informational pathways and biosignaling in the cell (Chap. 12) http://www. accessexcellence .org/AB/GG/ 1 Central Dogma of Molecular Biology Reverse transcription Replication Dogma: principle or set of principles laid down as incontrovertibly true Information Pathways and DNA - Chapter 24: GENES AND CHROMOSOMES -- Chromosomal Elements -- DNA supercoiling -- Structure of the chromosome 2 Chemical nature of nucleic acids Replication Reminder Chap. 8 3 Reminder Chap. 8 Molecular coding of protein sequence information •In transcription, one strand of double stranded DNA acts as molecular template for RNA synthesis •In translation, the triplets of nucleotides in mRNA are matched with corresponding amino acids via triplets of tRNA •Protein sequence determines its biological function 4 Part 1: Chromosomal Elements -> Genes, size and sequence composition Segments of DNA that code for functional polypeptide chains and RNA. Definition: gene In genes: presence of regulatory sequences -signals for beginning and end of genes (start, AUG; stop, UGA, UAA, UAG) -regulatory sequence for transcription, replication, recombination Possible to estimate the minimum overall size of genes (triplet codes for 1 aa) In E. coli (4.6 x 10 6 bp) -> Compact genome (2500 genes per mm) tot: ~4300 genes In Human (3 x 10 9 bp) -> Less compact (20 genes per mm) tot: 20500 genes ? Definition: chromosome • • Chromosome consists of one covalently connected DNA molecule and associated proteins. Chromosomes contribute to the compaction of the DNA molecules that are much longer than the cellular packages that contain them. – In viruses: Viral genomic DNA may be associated with capsid proteins – In bacteria: Prokaryotic DNA is associated with proteins in the nucleoid – In Eukayotes: Eukaryotic DNA is organized with proteins into a complex called the chromatin In bacteria: Generally one chromosome One copy of each gene (few repeats) Few interruptions of the coding sequence (DNA sequence is colinear with RNA sequence) Eukaryotic genes and chromosomes are much more complex! 5 (bp) How to estimate the length of a genome? 3.4 Å x number of bp (in the genome) = length of the genome Phage T4 (~170000 pb) HIV retrovirus (RNA genome of 9000 nt) Phage fd Parvovirus Bacteriophage phiX174 (~5400 pb) Human Poliovirus 6 DNA molecules in bacteria Dividing E.coli cell Plasmid DNA Size: 1.7 mm (4.6x10 6 bp) 2500 genes/mm 850 times the length of the E. coli cell Extra-chromosomal DNA -> Plasmids (2-10 x 10 4 bp) Resistant Genes (beta lactamases, see Chap. 20 box p. 779) 7 Eukaryotic DNA molecules are found in the nucleus , in mitochondria and in chloroplasts In Human 46 chromosomes in somatic cells 2 m of DNA per cell If 1014 cells, then 2x10 11 km (Earth-Sun dist. = 1.5 x 10 8 km) Karyotype 46 Chromosomes 24 different ones 8 Circular DNA duplexes! Mt DNA <20000 bp in mammals 1-10 copies of mt DNA/mitochondrion 2 x 10 5 -2 x 10 6 bp in plants Mt DNA codes for 5 % of mitochondria materials(mt rRNAs and tRNAs and proteins from inner membranes) Mt and Cp DNA are replicated before and during Mt and Cp division, when the cell divides. Cp DNA 1.2-1.6 x 10 5 bp Circular DNA duplexes! 9 Chapter 1 Higher Eukaryotes (plants, animals) Lower Eukaryotes (e.g. Yeast) Archaea (e.g. Methanobacterium) Bacteria Aerobic bacteria Cyanobacteria Mycoplasma (Parasitic bacteria that have lost some of their genes) Methanogen bacteria 10 Genomic sequencing timeline 11 Eukaryote genomes contain more DNA than do prokaryote genomes Relative size 1 25 20500 genes! 600 20500 genes in the human genome (2008 estimate)! DNA, Chromosomes, Genes, and Complexity • Despite the trends in the previous table, neither the total length of DNA, nor the number of chromosomes correlates strongly with the perceived complexity of the organisms – Amphibians have much more DNA than humans – Dogs and coyotes have 78 chromosomes in the diploid cell • • – Plants have more genes than humans The correlation between complexity and genome size is presently poor because most of eukaryotic DNA is not coding for proteins Recent experimental work by Craig Venter suggests that a minimal living organism (Mycoplasma) could get by with less than 400 genes 12 There is only 1.5% of DNA coding for proteins in human genome! Most Eukaryotic genes are interrupted! Non-translated DNA segments in genes are called intervening sequences, or introns . Coding segments are called exons . More intron sequence than exon sequence in most genes of higher eukaryotes Composition of the Human Genome • • Notice that only a small fraction (1.5 %) of the total genome encodes for proteins The biological significance of non-coding sequences is still not all clear – Some DNA regions directly participate in the regulation of gene expression (promoters, termination signals, etc) – Some DNA encodes for small regulatory RNA with poorly understood functions – Some DNA may be junk (pieces of unwanted genes, remnants of viral infections 13 Types of sequences in the human genome LINEs = Long Interspersed Elements Kind of parasites that contribute to evolution of the genome SINEs = Short Interspersed Elements SSR = Simple Sequence Repeats Satellite DNA SD = Segmental Duplication Transposons • DNA sequence is not completely static • Some sequences, called transposons, can move around within the genome of a single cell • The ends of transposons contain terminal repeats that hybridize with the complementary regions of the target DNA during insertion Chapter 25 14 Snapshot of the human genome Eukaryotic Chromosomes Highly repetitive sequences Simple sequence repeats (SSR) Or Satellite DNA 8 %, 10bp x 106 30 % Genes (set of exons ) and intervening sequences ( introns ) in centromeres 5-10bp in tandem copies ( Euk. sup.) 130bp AT rich sequences (yeast) in telomeres 5’(TxGy)n with (x,y) = 1-4 n= 1500 in mammals 45% transposons (moderately repetitive sequences) 200-300 bp x 10 3 Related to transposable elements (transposons )-> Chap 25 Eg . 10 5-10 6 Alu copies in Human genome 17% ??? Small regulatory RNAs ? How to build an artificial chromosome? Need telomeres, centromere , sequences for initiation of replication ORI -> See Chap 9 ( YACs (Yeast AC) and HACs (Human AC) 15 G quartet Structure of telomeres (Greek telos, “end) Part 2: DNA Supercoiling • DNA in the cell must be organized to allow: – Packing of large DNA molecules within the cells – Access of proteins to read the information in DNA sequence • There are several levels of organization, one of which is the supercoiling of the double-stranded DNA helix 16 What is supercoiling? Relaxed state Strained state (no net bending of the (DNA super helix = DNA axis upon itself) DNA supercoil ) Phone cord = coil Supercoil = coil of a coil Supercoiling is a physical manifestation of structural strain 17 Supercoiling Underwinding Separation of strands of a helical structure results in supercoiling ! => Replication and transcription processes will be affected by DNA supercoiling Most cellular DNA is underwound Case study: circular plasmid DNA No supercoiling Tightly supercoiled Supercoiling is an intrinsic property of DNA. This process is highly regulated by cells. 18 For closed circular B DNA -> 10.5 bp / turn in relaxed state In cellular DNA structural strain results from underwinding the DNA in the closed circle (less helical turns than expected for B DNA) 84 pb (8 turns 10.5 bp/ turn) (10.5 bp/turn -> 12 bp/turn) -> Thermodynamic destabilization of the helix structure To accommodate this: (1) formation of a supercoil (2) separation of strands (1) requires less energy than (2) but, (2) can give access to genetic information Number of helical turns can not change without breakage of one strand (require enzymatic process) (2) 84 pb (7 turns 12 bp/ turn) (1) DNA strain is a form of stored energy! Topology: Study of the properties of an object that do not change under continuous deformations. Continuous deformations: thermal motion/ interactions with other molecules Discontinuous deformations: imply strands breakage Topological bond Linking Number: Lk (Integer) (+) It is a topological property Lk = 4 For a relaxed circular DNA: (tot Nb of bp) Lk0 = ____________ (Nb of bp/turn) ( Lk0 is equal to the number of turns) 19 DNA underwinding is defined in terms of changes ΔLk in the Lk number If Lk0 for the relaxed DNA and Lk for the strained DNA ΔLk =Lk -Lk0 =N Specific linking difference or superhelical density ( σ) (independent of length) σ= ΔLk ____ Lk0 For cellular DNA σ = -0.05, -0.07 If sign of ΔLk or σ is negative Underwinding -> negative supercoil If sign of ΔLk or σ is positive Overwinding -> positive supercoil Supercoiling is not random It is prescribed by torsional strain imparted to the DNA by ΔLk relative to B DNA. Topoisomers: two forms of a given circular DNA that differs only in topological property Lk 20 Topological Geometrical { Twist: local twisting (spatial relationship of adjacent bp) Writhe: measure of the coiling of the helical axis Ribbon model for illustrating twist (T) and writhe (W) Twist and writhe are geometrical properties Lk = Tw + Wr 21 L=T+W Promotion of cruciform by DNA underwinding Underwinding can also promote formation of short stretches Z DNA (left handed) 22 DNA supercoiling is regulated by topoisomerases Increase or decrease the extend of DNA underwinding Type I topo: break one strand, rotate the other by 360° and rejoin the broken ends: change Lk by increment of 1 Type II topo: break two strands and change Lk by increment of 2 In Prokaryotes (e.g E. Coli) Type I: topoisomerases I and III -> relax DNA by increasing Lk Type II: topoisomerase II = DNA gyrase -> decreases Lk In Eukaryotes Type I: topoisomerases I and III -> relax DNA by increasing Lk Type II: topoisomerases II α and IIβ -> relax + and - supercoils but do not underwind DNA (other mechanism involving the nucleosome) Change in linking catalyzed by topoisomerases Visualization of DNA topoisomers generated with bacterial type I topoisomerase 23 Type I topoisomerase Tyr Type I topo: breaks one strand, rotates the other by 360 ° and ligates the broken ends: this changes Lk by increment of 1 David S. Goodsell 24 25 Mechanism of topoisomerase II or DNA gyrase (Prokaryote) Eukaryotic Type IIA topoisomerase Can relax both positive and negative supercoils David S. Goodsell 26 Mechanism for the alteration of Lk by Eukaryotic type IIA topoisomerases This process requires 2 ATP The chemistry is similar to the one of bacterial type I topoisomerase Plectonemic DNA supercoiling Supercoil is right handed in negative supercoiled DNA Extended and narrow with multiple branches structures Length of the supercoil corresponds to 40 % of the length of DNA Plectonemic DNA is observed in the laboratory in isolated DNA 27 Plectonemic and solenoidal DNA supercoiling Solenoidal supercoiling (account for the extraordinary compaction of DNA in cells) Left handed solenoid Both structures are interconvertible (can be taken by the same segment of underwound DNA) In solution, plectonemic more stable than solenoidal In cells, solenoidal can be stabilized by proteins. Solenoidal DNA is the form found in chromatin (chromosomal material) Structure of chromosomes 28 => Reminder: Chromosome = nucleic acid molecule repository of the genetic information in virus, bacteria cell or organelles. -- In Eukaryotes, they are sharply defined bodies just before & during mitosis. => In non dividing cell, the chromosomal material is called chromatin. -- It is amorphous and is randomly dispersed in the nucleus. -- Chromatin consists of fibers containing DNA and protein (MW 1:1) and a small amount of RNA. -- DNA tightly associated with histones form structural units called nucleosomes. -- Other proteins (involved in the regulation of genes) are found in chromatin Change in chromosome structure during the eukaryotic cell cycle 29 The nucleosome beads on a “Beads-on-a-string” arrangement are histones complexes bound to DNA. Electron micrograph of nucleosomes Nucleosome core= 8 histones 2 copies each of H2a, H2b, H3 & H4 ~200 bp DNA, 146 bp tightly bound to the histone core (discovered by partial digestion of chromatin with nucleases (Chap. 25)) H1 binds to the ~ 50 bp DNA linker 30 Histones are small basic proteins of MW between 11000 and 21000 -> H3 and H4 sequences are very conserved in all eukaryotes (suggest a strict conservation of functions (2aas ≠ between peas and cows, 8aas ≠ between yeast and humans) -> H1, H2a and H2b are less conserved -> Histones are modified proteins (by methylation, ADP ribosylation , phosphorylation and acetylation). Modifications affect histones net electric charge, shape and properties as well as structure function properties of chromatin (see Box 24.2 and Chap. 28). Crystallographic X-ray structure Luger, K., Mader, A. W., Richmond, R. K., Sargent , D. F., Richmond, T. J. (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389 pp. 251 31 How is eukaryotic DNA underwound? ~ -1.0 +1 ΔLK= +1 - 2 = -1 Solenoidal wrapping of DNA around histone core requires the removal of about one helical turn. This is achieved by topoisomerases II that relaxes positive DNA supercoiling. This has been demonstrated in vitro with purified histones and circular DNA. Inhibitors of topo II can rapidly kill dividing cells. In chemotherapy, some drugs are used to inhibit topo II by allowing the enzyme to promoting strand breaking but not the resealing of the breaks. David S. Goodsell A-T pairs (minor grove) The sequence of the bound DNA affects its binding to histones . The binding is not random (requires an abundance of A-T pairs in the minor grove of the helix where it is in contact with histones ). 32 Other proteins are required for the precise positioning of nucleosome cores on DNA. Some proteins have been discovered to bind to a specific DNA sequence and favor nucleosome formation nearby. This can play an important role in Eukaryote genes expression. The chromatin is remodelled by acetylation and nucleosomal displacements in order to favor transcription (See chapter 28). David S. Goodsell Higher order structures of chromatin DNA wrapping around nucleosome core -> 7 fold compaction 30 nm fibers -> 100 fold compaction -> require one H1 per nucleosome core This type of organization does not extend to the entire chromosome but is punctuated by regions bound by sequence specific DNA-binding proteins. -> Depend on the transcriptional activity of DNA: regions that are transcribed are in a less ordered state with no H1 (or very little). 33 Higher levels of structure --Less well understood --Certain regions seem to be associated to a nuclear scaffold --The scaffold associated regions are separated by loops of DNA with 20000 to 100000 bp. --Set of related genes seem to be clustered within these loops (e.g.: histone coding genes in Drosophila) --The scaffold seems to be rich in H1 and topo II DNA Scaffold like structure The overall compaction of the DNA within Eukaryotic chromosomes is greater than 10000 fold ! Compaction is likely to involve coils upon coils upon coils upon coils… Model 34 Condensed chromosome structures are maintained by SMC proteins SMC Proteins: Structural Maintenance of Chromosomes Proteins Cohesins and condensins Cohesins link sister chromatids after replication and keep them together during cell division (assure good chromosome segregation). 35 DNA “safety pin ” Essential for the condensation of the chromosomes as cell enter into mitosis Change in chromosome structure during the eukaryotic cell cycle 36 Bacterial DNA is compacted in a structure called nucleoid Nucleoid --> Not very well known --> The DNA appears to be attached at the inner surface of the plasma membrane --> Scaffold-like structure appears to organize the circular chromosome into a series of looped domains as for chromatin but, no local organization like nucleosomes Histone like proteins (Two subunits protein HU, 19000d) -> do not seem to create very stable structure with DNA (bind and dissociate within minutes) Structure of the nucleoid Bacterial Chromosome Contain SMC proteins too! => The bacterial chromosome is a relatively dynamic structure (need of access to genetic information as bacterial cell division cycle can be very short compared to eukaryotic cells) 37 Conclusion -> Prokaryotes and Eukaryotes have selected 2 different ways for packaging DNA that reflect their different evolutionary pathways Eukaryotes Prokaryotes The results of an evolution towards miniaturized living systems, with high rates of multiplication (replication is 10 times faster than in Eukaryotes), and high metabolic rates (meaning higher proportion of the genome is transcribed and replicated) The result of an evolution towards a greater complexity Note that Prokaryotes and Eukaryotes have a common ancestor LUCA (Last Universal Common Ancestor) 38 ...
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