10/23/2011. Quiz Oct Overview: Life s Operating Instructions DNA

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1 hapter 6 DN: he Molecular Basis of Inheritance Quiz Oct In the protein coupled receptor - IP3 pathway, the α subunit of the -protein activates what molecule? 2. In the protein coupled receptor - cmp pathway, the α subunit of the -protein activates what molecule? 3. (2 pts) What are two ways cell signaling pathways are regulated? 4. What is the function of protein kinase Overview: Life s Operating Instructions Deoxyribonucleic acid, or DN, the substance of inheritance, is the most celebrated molecule of our time Hereditary information is encoded in DN and reproduced in all cells of the body his DN program directs the development of biochemical, anatomical, physiological, and (to some extent) behavioral traits DN Deoxyribonucleic acid DN he blueprint to making proteins!!! hromosomes located inside the nucleus contains long coiled s of DN Figure 6.3 Phage head ail sheath ail fiber DN Bacterial cell 00 nm

2 0/23/20 Hershey and hase Experiments In 952, lfred Hershey and Martha hase performed experiments showing that DN is the genetic material of a phage known as 2 o determine this, they designed an experiment showing that only one of the two components of 2 (DN or protein) enters an E. coli cell during infection hey concluded that the injected DN of the phage provides the genetic information nimation: Hershey-hase Experiment Right-click slide / select Play Figure 6.4- Figure EXPERIMEN Phage EXPERIMEN protein Phage Bacterial cell Batch : sulfur (35S) protein Empty protein shell Bacterial cell Batch : sulfur (35S) DN DN DN Phage DN DN Batch 2: phosphorus (32P) Batch 2: phosphorus (32P) Figure DN s Discovery EXPERIMEN Phage protein Empty protein shell Radioactivity (phage protein) in liquid Bacterial cell Batch : sulfur (35S) DN Phage DN entrifuge Pellet (bacterial cells and contents) DN Batch 2: phosphorus (32P) entrifuge Pellet Radioactivity (phage DN) in pellet Watson and rick Rosalind Franklin 2

3 he Players rick: Ph.D. student at ambridge in England working on X-ray rystallography of the protein hemoglobin Watson: Young merican scientist visiting the lab to do some work on a protein Both were interested in unraveling the secret of DN s structure it was not what they were supposed to be working on Wilkins: Working on DN structure, had crystallized DN fibers Franklin: Working at the same university as Wilkins, just down the hall. Did the X-ray rystallography on Wilkins DN fibers Linus Pauling: discovered the three dimensional structure of proteins know as alpha helixes hargaff: Discovered that = and = denine levels always equal thymine levels, uanine levels always equal cytosine Franklin gave a talk describing her work with the X-Ray rystallography, Watson attended but he was not the crystallographer and did not see the implications of her work Watson and rick met with Wilkins and he shared Franklin s work with both of them (without her permission or knowledge) Watson and rick put all the pieces of information together. hey built models to help them come up with the structure. hey knew it was a race so they published a one page article in Nature with their ideas they performed no experiments but were able to see the big picture rick, Watson and Wilkins received the Nobel Prize for their work. Rosalind received no credit until much later. She died before the Nobel Prize, the prize is not awarded after a person has died Fig. 4.4 Nucleotide Structure nimation: DN and RN Structure Rightclick slide / select Play 3

4 Figure 6.5 Sugar phosphate backbone end Nitrogenous bases hymine () denine () ytosine () Phosphate uanine () Sugar (deoxyribose) DN nucleotide end Nitrogenous base nimation: DN Double Helix Rightclick slide / select Play DN Structure Fig Nucleotides that build DN have: One phosphate (P has three) One sugar = deoxyribose One base. he nucleotides vary in the type of base there are four different bases in DN: denine (), hymine (), uanine (), ytosine () here is a 5 end and a 3 end 4

5 Bonds he sugars and phosphates link together by covalent bonds to form the rail on the outside = phosphodiester linkage. he sugars are covalently bound to a base he complementary bases are attracted to each other by hydrogen bonds Double Helix wo s bonded together by hydrogen bonds between the bases = weak bonds Each has nucleotides bonded together covalently by the phosphate and the sugar Base pairs are two nucleotides, one on each complementary of a DN molecule 5

6 Base Pairs Figure 6.8 he bases pair up in a specific manner: denine () pairs with hymine () uanine () pairs with ytosine () Purines: denine and uanine Pyrimidines: hymine and ytosine Sugar denine () Sugar hymine () Sugar Sugar uanine () ytosine () he bonds between the sugars and phosphates are Remember that on one : he base is covalently bonded to the sugar, which is covalently bonded to the phosphate Between the two s the bases are bonded together by hydrogen bond. Peptide 2. Phosphodiester 3. Hydrogen 4. Ionic he bonds between the bases are denine pairs with. Peptide 2. Phosphodiester 3. Hydrogen 4. Ionic. hymine 2. uanine 3. ytosine 6

7 uanine pairs with he bases are bound to. hymine 2. denine 3. ytosine. Sugars 2. Phosphates he bases are bound to the sugar by this kind of bond he sugar in DN is. ovalent 2. Phosphodiester 3. Hydrogen 4. Ionic. Ribose 2. Deoxyribose 3. lucose 4. ellulose Many proteins work together in DN replication and repair he relationship between structure and function is manifest in the double helix Watson and rick noted that the specific base pairing suggested a possible copying mechanism for genetic material DN Replication When the structure of DN was worked out it became apparent how it happens It is semiconservative replication Each of DN is the template for building new complementary s 7

8 he Basic Principle: Base Pairing to a emplate Strand Since the two s of DN are complementary, each acts as a template for building a new in replication In DN replication, the parent molecule unwinds, and two new daughter s are built based on base-pairing rules Figure 6.9- (a) Parent molecule nimation: DN Replication Overview Right-click slide / select Play Figure Figure (a) Parent molecule (b) Separation of s (a) Parent molecule (b) Separation of s (c) Daughter DN molecules, each consisting of one parental and one new 8

9 Semiconservative model Figure 6.0 Parent cell First replication Second replication Watson and rick s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old (derived or conserved from the parent molecule) and one newly made (a) onservative model (b) Semiconservative model ompeting models were the conservative model (the two parent s rejoin) and the dispersive model (each is a mix of old and new) (c) Dispersive model Fig. 4.3 DN Replication: loser Look he copying of DN is remarkable in its speed and accuracy More than a dozen enzymes and other proteins participate in DN replication etting Started Replication begins at particular sites called origins of replication, where the two DN s are separated, opening up a replication bubble eukaryotic chromosome may have hundreds or even thousands of origins of replication Replication proceeds in both directions from each origin, until the entire molecule is copied t the end of each replication bubble is a replication fork, a Y-shaped region where new DN s are elongating nimation: Origins of Replication Right-click slide / select Play 9

10 Figure 6.2 (a) Origin of replication in an E. coli cell Origin of replication Doubleed DN molecule wo daughter DN molecules Parental (template) Replication bubble Daughter (new) Replication fork (b) Origins of replication in a eukaryotic cell Origin of replication Parental (template) Bubble Double-ed DN molecule Daughter (new) Replication fork wo daughter DN molecules DN DN Replication. n enzyme, helicase, unwinds the DN molecule and breaks the hydrogen bonds between the base pairs 2. Single- binding proteins (helix destabilizing proteins) bind to each and keep them from reforming the double helix 3. opoisomerases (DN gyrase) produce breaks in the DN molecule to relieve the stress of unwinding, then they also repair these breaks. 0.5 m 0.25 m Figure 6.3 DN Replication opoisomerase Helicase Primase Single- binding proteins RN primer Now the complementary needs to be built: 4. Enzymes called DN polymerases build the new complementary by adding new nucleotides to the 3 end which pair with the old DN. 5. But DN polymerase can not start the process. primer of RN bases is first built for the complementary. 6. n enzyme called primase adds the RN bases, then DN polymerase can take over and keep building the complementary. 7. he primer is replaced by DN bases DN DN Replication DN DN Replication 8. DN polymerase builds the new complementary from the 5 end to the 3, by adding the nucleotides to the 3 end = leading But the other also needs to be replicated but it can only build new s by adding to the 3 end 9. he other = lagging, is build in short stretches going from 5 to 3 0. he short s being built are called Okazaki fragments. DN ligase join the Okazaki fragments 0

11 Synthesizing a New DN Strand Enzymes called DN polymerases catalyze the elongation of new DN at a replication fork Most DN polymerases require a primer and a DN template he rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells Synthesizing a New DN Strand Each nucleotide that is added to a growing DN is a nucleoside triphosphate dp supplies adenine to DN and is similar to the P of energy metabolism he difference is in their sugars: dp has deoxyribose while P has ribose s each monomer of dp joins the DN, it loses two phosphate groups as a molecule of pyrophosphate Energy to power building complementary he incoming nucleotides have three phosphates, only one is used to bond to the sugar molecule Figure 6.4 Sugar Phosphate New Base emplate he energy needed to build the new DN comes from taking the other two phosphates off. OH DN polymerase he energy gained from breaking the bonds is used to build the new bond Nucleoside triphosphate P P i Pyrophosphate 2 P i OH ntiparallel Elongation ntiparallel Elongation he antiparallel structure of the double helix affects replication DN polymerases add nucleotides only to the free end of a growing ; therefore, a new DN can elongate only in the to direction long one template of DN, the DN polymerase synthesizes a leading continuously, moving toward the replication fork

12 ntiparallel Elongation o elongate the other new, called the lagging, DN polymerase must work in the direction away from the replication fork he lagging is synthesized as a series of segments called Okazaki fragments, which are joined together by DN ligase nimation: Leading Strand Rightclick slide / select Play ntiparallel Elongation Figure 6.6a Leading Overview Origin of replication Lagging Lagging 2 Overall directions of replication Leading nimation: Lagging Strand Rightclick slide / select Play Figure 6.6b- Figure 6.6b-2 emplate emplate RN primer for fragment 2

13 Figure 6.6b-3 Figure 6.6b-4 emplate RN primer for fragment emplate RN primer for fragment Okazaki fragment RN primer for fragment 2 Okazaki fragment 2 Okazaki fragment 2 Figure 6.6b-5 Figure 6.6b-6 emplate RN primer for fragment emplate RN primer for fragment RN primer for fragment 2 Okazaki fragment 2 Okazaki fragment 2 RN primer for fragment 2 Okazaki fragment 2 Okazaki fragment Overall direction of replication Figure 6.7 Figure 6.7a Leading Overview Origin of replication Lagging Overview Leading Origin of replication Lagging Leading Lagging Overall directions of replication Leading Lagging Overall directions of replication Leading DN pol III Parental DN Primer Primase DN pol III 4 Lagging DN pol I DN ligase 3 2 Parental DN Leading DN pol III Primer Primase 3

14 Figure 6.7b Leading Overview Origin of replication Lagging he DN Replication omplex Leading Lagging Overall directions of replication Leading he proteins that participate in DN replication form a large complex, a DN replication machine Primer DN pol III Lagging DN pol I DN ligase he DN replication machine may be stationary during the replication process Recent studies support a model in which DN polymerase molecules reel in parental DN and extrude newly made daughter DN molecules Figure 6.8 DN pol III Parental DN Leading onnecting protein Helicase DN pol III Lagging Lagging template nimation: DN Replication Review Right-click slide / select Play Replicating the Ends of DN Molecules Youube - DN Replication Process Youube - DN Replication (Very realistic 3D animation) Limitations of DN polymerase create problems for the linear DN of eukaryotic chromosomes he usual replication machinery provides no way to complete the ends, so repeated rounds of replication produce shorter DN molecules with uneven ends his is not a problem for prokaryotes, most of which have circular chromosomes 4

15 Replication at end of DN t the end of the DN a small portion of the is not replicated Figure 6.20 Ends of parental DN s Lagging Last fragment RN primer Leading Lagging Next-to-last fragment So we don t lose important genetic information, DN s have non-coding end caps Parental Removal of primers and replacement with DN where a end is available Second round of replication hese end caps are called telomeres New leading New lagging Further rounds of replication Shorter and shorter daughter molecules Figure 6.20a Figure 6.20b Ends of parental DN s Leading Lagging Second round of replication Last fragment Next-to-last fragment Lagging RN primer New leading Parental Removal of primers and replacement with DN where a end is available New lagging Further rounds of replication Shorter and shorter daughter molecules elomeres Figure 6.2 Eukaryotic chromosomal DN molecules have special nucleotide sequences at their ends called telomeres elomeres do not prevent the shortening of DN molecules, but they do postpone the erosion of genes near the ends of DN molecules It has been proposed that the shortening of telomeres is connected to aging m 5

16 Replication at end of DN Fig elomerase build the telomeres. Embryos have high telomerase activity, as you age you lose this activity. ancer cells have telomerase activity Fig Mistakes repair mechanisms Before a cell can divide, it must make a complete copy of itself here are millions of bases that need to be added to the DN s many chances for something to go wrong Enzymes will take out the wrong nucleotide and replace it with the correct one Proofreading and Repairing DN DN polymerases proofread newly made DN, replacing any incorrect nucleotides In mismatch repair of DN, repair enzymes correct errors in base pairing DN can be damaged by exposure to harmful chemical or physical agents such as cigarette smoke and X-rays; it can also undergo spontaneous changes In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DN Repair Mechanisms Photorepair UV light can cause thymine dimers to occur. Photolyase uses visible light to break dimer Excision Repair mismatched pairs are recognized and removed, DN polymerase builds the new correct pairing 6

17 Fig Fig Figure 6.9 Evolutionary Significance of ltered DN Nucleotides Nuclease DN polymerase DN ligase Error rate after proofreading repair is low but not zero Sequence changes may become permanent and can be passed on to the next generation hese changes (mutations) are the source of the genetic variation upon which natural selection operates auses of Mutations Random error sometimes things just go wrong. Results of Mutations few things can happen if DN mutates before the cell replicates: Mutagens chemicals that damage the DN and cause mutations in replication igarette smoke Sunlight Many chemicals (benzene) Enzymes can repair the damage Or he cell may commit suicide (apoptosis) Or he cell may replicate and the mutation becomes permanent 7

18 he enzyme that unwinds the DN molecule and breaks the hydrogen bonds between the base pairs. DN polymerases 2. Helicase 3. opoisomerases 7% would pair with % 8% DN polymerases Helicase opoisomerases hromosome consists of a DN molecule packed together with proteins he bacterial chromosome is a double-ed, circular DN molecule associated with a small amount of protein Eukaryotic chromosomes have linear DN molecules associated with a large amount of protein In a bacterium, the DN is supercoiled and found in a region of the cell called the nucleoid hromosome consists of a DN molecule packed together with proteins hromatin, a complex of DN and protein, is found in the nucleus of eukaryotic cells hromosomes fit into the nucleus through an elaborate, multilevel system of packing Figure 6.22a DN double helix (2 nm in diameter) Nucleosome (0 nm in diameter) DN, the double helix Histones Histones Histone tail H Nucleosomes, or beads on a string (0-nm fiber) nimation: DN Packing Right-click slide / select Play 8

19 Figure 6.22b hromatid (700 nm) hromosome consists of a DN molecule packed together with proteins 30-nm fiber hromatin undergoes changes in packing during the cell cycle 30-nm fiber Loops Scaffold 300-nm fiber t interphase, some chromatin is organized into a 0-nm fiber, but much is compacted into a 30-nm fiber, through folding and looping Looped domains (300-nm fiber) Replicated chromosome (,400 nm) Metaphase chromosome hough interphase chromosomes are not highly condensed, they still occupy specific restricted regions in the nucleus Figure 6.23 hromosome consists of a DN molecule packed together with proteins Most chromatin is loosely packed in the nucleus during interphase and condenses prior to mitosis Loosely packed chromatin is called euchromatin During interphase a few regions of chromatin (centromeres and telomeres) are highly condensed into heterochromatin 5 m Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions Important oncepts DN wrapping around proteins Know the vocabulary in this lecture Structure of DN and their nucleotides he four bases, and which are paired together Be able to recognize the four base structures Know which bases are purines and Pyrimidines ype of bonds/linkages Be able to draw DN for me (you can use S and P for sugar and phosphate, for bases, 5 and 3 ) 9

20 Important oncepts Be able to describe how is DN replicated Semiconservative replication Steps omplementary pairing Direction of building the complementary pair he role of helicase, Single- binding proteins opoisomerases, DN polymerases, DN ligase Understand how the leading is build vs how the lagging is built, know what Okazaki fragments are, Important oncepts Know what telomers and telomerases are What supplies the energy to be used to build the new Be able to identify correctly paired bases and incorrectly paired bases Know the repair mechanisms for DN 20