Lecture 11 Topic X. Genetic Material DNA _ RNA

Lecture 11 Topic X. Genetic Material DNA _ RNA - BIS101-01:...

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Unformatted text preview: BIS101-01: Genes and Gene Expression Topic X. Genetic Material: DNA & RNA 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 1 Last lecture: Topic VII. Genetic Analysis of Bacteria VII. Genetic Analysis of Bacteria A. 1. 2. 3. Genotype analysis Lethal and conditional mutants Auxotrophs and prototrophs Replica plating (Handout #12) A. B. 1. 2. 3. 4. Transformation Conjugation F factor Hfr strains Interrupted mating Hfr x FGene order (Handout #13) Map distances in recombination units F’: partial diploid BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 2 5. 03/29/10 Last lecture: Topic VII. Genetic Analysis of Bacteria A. 1. 2. 3. 4. Transduction Bacteriophage structure Life cycle of virulent phage (Handout #14) Generalized transduction Mapping Cotransduction (Handout #15) 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 3 This lecture: Topic X. Genetic Material- DNA and RNA A. DNA as genetic material 1. Griffith experiment 2. Avery, MacLeod, and McCarty experiment 3. Hershey and Chase experiment B. Chemical composition of DNA/RNA 1. Nucleotides (Handout #17) 2. Phosphodiester bond (Handout #18) C. Double Helix Model 1. X-ray diffraction (Franklin and Wilkens) (Handout #19) 2. Chargaff's rules ((Handout #19) 3. Base pairing (Handout #20) 4. Watson and Crick model (Handout #19) D. RNA structure 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 4 Characteristics of genetic material ­ In order to serve as genetic material, DNA must In have the following four characteristics: have q q q q 1. Chemical stability under a variety of environmental conditions 1. Chemical (e.g., 0°C to 100°C and 0.01M salt to about 4 to 5M salt). DNA can be detected in a 10,000 year old mammoth or 400,000 years old plants. plants. 2. DNA must be capable of self-replication (the ability to pass 2. self-replication copies of itself from one generation to the next). copies 3. Must have the potential to store and transfer information. Triplet 3. store Triplet codons are used to code for the sequence of amino acids in proteins. codons 4. Genetic material must be mutable, that is, capable of undergoing 4. rare chemical changes. Mutations provide the genetic variability for Mutations natural selection. BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 5 2/16/10 Genetic material Proof that DNA is the genetic Proof material — Three key experiments. experiments. 2/16/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 6 First key experiment A. DNA as genetic material Three key experiments related to this idea that DNA is the genetic material. Three 1. Frederick Griffith, 1928 1. Studied the bacterium Streptococcus pneumoniae Studied Streptococcus -causes pneumonia in humans Two different strains characterized by their surface phenotype: Two phenotype R strain cells (Type IIR) rough surface nonvirulent S stain cells (Type IIIS) smooth surface virulent Griffith studied the effect of these strains on mice -injected the bacteria into the mice. -made the following observations: 1. R bacteria mouse lives R iis nonvirulent s 1. 2. S bacteria mouse dies S is virulent 3. heat-killed S bacteria mouse lives Cell debris 3. 4. heat-killed S and live R mouse dies -can recover virulent S from and mouse mouse 03/29/10 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 7 Transforming R cells into S cells 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 8 Griffith’s Conclusions Griffith’s Griffith defined the process as follows: S-cell component live R cells --------------------------------------------> live S cells Transformation Conclusions: Live R cells transformed to S-type cells. Process of bacterial transformation. R cells become virulent by picking up virulent S DNA. All occurred in the mouse so this was an in vivo experiment. 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 9 Second key experiment 2. Avery, MacLeod and McCarty, 1944 Did the same experiment of transforming nonvirulent into virulent Did strains but did the experiment in vitro. in Transformed live R -->live S in a test tube. Next, they fractionated various components of the S cells. They looked for the S cells components that converted the R cells They to S cells. to S cell component Result Polysaccharides + R ---------> R Polysaccharides llipids + R ---------> ipids R proteins + R ---------> R proteins RNA/DNA + R ---------> R&S RNA/DNA RNA/DNA + RNase + R --------> R&S RNA/DNA (Ribonuclease: degrades RNA) RNA/DNA + DNase + R --------> RNA/DNA 03/29/10 R 10 (Deoxyribonuclease: degrades DNA) BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression DNA is the transforming agent 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 11 Conclusion ­ DNA was the transforming material. DNA determined the pathogenic character, therefore DNA DNA was the genetic material. was At the time of the experiment, this was a surprise because At DNA was not known to be part of Streptococcus pneumoniae. pneumoniae 03/29/10 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 12 Third key experiment 3. Hershey and Chase, 1952 Post WW II, scientists had access to radioisotopes as tracers. Post Scientists were convinced that DNA could “transform” bacteria. Scientists Hershey and Chase asked the question: Hershey Is DNA the genetic material? Is -worked with a bacteriophage: T2 phage (virus): -labeled T2 with two different radioisotopes: -labeled 35S-T2 phages (labeled proteins; sulfur) 35 32P-T2 phages (labeled DNA; phosphate= major P-T2 component of backbone) component Experiment: infect bacteria with the labeled T2 phages, this directs more Experiment: phage to be made, ask the question: What is the cellular component that’s doing this: What (1) DNA ? (as suggested by Avery, MacLeod and McCarty), or (1) DNA (2) Protein? Protein? 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 13 3. Continued: Hershey and Chase Experiment Radiolabeled phage: Phage with 35S labeling: this will “tag” protein Phage 35 Phage with 32P labeling: this will “tag” DNA Phage 32 Infect E. coli with phage. Infect E. Put in a blender and shear off the “phage ghosts”: This phage material is what didn’t get inside the bacterial cell. Idea was that whatever gets inside the cell is directing a change in the bacterial cell, i.e., is the “transforming principle.” the Results: Phage with 35S llabeling: most of radioactivity in phage ghosts so Phage 35 abeling: outside. outside. Phage with 32P llabeling: most of radioactivity inside cell, passed on Phage 32 abeling: to phage progeny. to 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 14 The phage genetic material is DNA 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 15 Hershey and Chase Experiment (1951) Alfred Hershey joined the APG and used 32P labeled 32 phage DNA and 35S 35 labelled phage coat labelled proteins to show that only 32P was 32 was transferred from parental phage to progeny phage. This experiment finally laid to rest the protein/DNA debate and demonstrated the importance of the Avery et al. discovery et 2/16/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 16 Summary Hershey and Chase experiment: showed using radioactivity that it was the DNA component, and not the protein component, that had entered the bacterial cell and been passed onto the progeny. DNA must be directing genetic change or at least carried that information. that Conclusions: DNA is the genetic material -almost universal -sometimes RNA is genetic material (RNA viruses). 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 17 B. Chemical composition of DNA/RNA 1. Nucleotides: DNA and RNA are polymers of 1. nucleotides. nucleotides -Collectively called “nucleic acids”. Some terms: nucleosides: sugar + base nucleotides: sugar + phosphate + base Nucleic acids are polymers of nucleotides, called the Nucleic “polynucleotide chain” “polynucleotide -usually illustrated by line drawings: horizontal line: represents backbone (sugar + phosphate) vertical line: represents bases coming off the backbone Handout #17 - Basic structures of the nucleotides. 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 18 Basic structure of the nucleotides Handout #17 - Basic structures of the nucleotides. a. Bases: Two kinds of bases: purines and pyrimidines (1) Purine: 2-membered ring of carbons and nitrogens. Two specific purines: adenine = A guanine = G Purines are planar molecules, containing N. Also called the “nitrogenous bases” of DNA/RNA. (2) Pyrimidines: also ring structure of carbons and nitrogens. Three specific pyrimidines: cytosine = C thymine = T These two differ in the methyl group group uracil = U (U lacks methyl group) Note: Thymine found only in DNA. Uracil found only in RNA. 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression Other three (A, G, C) found in both DNA/RNA. 19 The Nitrogenous Bases DNA RNA 2/16/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 20 b. Sugars Basic sugar is ribose, a 5-C sugar (HO #17 middle, far right). -need to know numbering of carbons: 1’ to 5’ C. Two ribose sugars of interest to us: Ribose (left) Deoxyribose (middle) Q. Difference between these two? Q. Difference No hydroxyl group on the 2’C of deoxyribose. No (Note the presence of the 2’ -OH on ribose). (Note and Ribose is only found in RNA, Deoxyribose is only found in DNA. Some shorthand: Example: Ribose We typically draw a vertical line to indicate OH groups and will leave off the hydrogens (H) altogether (middle right). leave 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 21 Nucleotides: The Sugars O - P O N N O H N H N N H - P O N N 5’ H 4’ 3’ O O C H O H N H N N H 5’ H O O C H O 1’ 2’ 4’ 3’ O - 1’ 2’ H O - OH O P O P O O O Deoxyribose O Ribose 2/16/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 22 c. Nucleotide: Base + sugar + phosphate How are they all connected? Base is connected to the sugar molecule at the 1’ C by a C-N bond. Phosphate can connect to 5-C sugar at a number of places (2’, 3’, 5’) We are interested in 3’ and 5’ phosphates. Handout #17: Two examples on bottom Two Handout C-N bond to 1’ C; phosphate group at 5’ C position. #1) Deoxcytidine 5’-monophosphate Deoxy = deoxyribose sugar cytidine= cytosine is the base 5’-monophosphate= one phosphate at 5’ C of sugar. #2) Adenosine 5’-monophosphate Not deoxy = so a ribose sugar adenosine= adenine is the base 5’-monophosphate= one phosphate at 5’ C of sugar. 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 23 Structure of the four DNA nucleotides 03/29/10 BIS101­001, Spring 2010—Genes and Gene Expression, S. O’Neill ©2009 BIS101­001, Spring 2010—Genes and Gene Expression, S. O’Neill 24 Nucleotides: The Phosphates - P O O - O P O O O O P O O P O O C O N H N H O N - O H P O O N H N H N N H O - P O O N H N H N N H H O N N H O H O H C H O N C H O N OH H OH H OH H Deoxyadenosine Monophosphate 2’ position if OH, then Adenosine Monophosphate Deoxyadenosine Diphosphate 2’ position - if OH, then Adenosine Diphosphate Deoxyadenosine Triphosphate 2’ position - if OH, then Adenosine Triphosphate 2/16/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 25 d. Polynucleotide chain: string of connected nucleotides 1. How is each nucleotide in the chain connected? 1. How Connected with a phosphate group. Bonds formed between a 5’and 3’ carbon on sugar: Bonds -called a “phophodiester bond:” -called A phosphate group forms a bridge between -OH groups on two phosphate adjacent sugar residues. adjacent Look at first unit of chain: Look -phosphate attached to 5’ C Next unit down: -connected to above unit by phosphate, now attached -connected to 3’ C of first deoxynucleotide, and to 5’ at next unit. to Phophodiester bond -creates the “sugar-phosphate” backbone. -very stable bond so the backbone is stable. At end of chain, there is a 3’ end with a hydroxyl (OH) group. 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 26 Reaction catalyzed by DNA polymerase 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 27 2. This is a short polynucleotide chain: 2. By convention: By -the nucleotide sequence is defined by -the the bases. -the sequence is listed 5’ to 3’. -the Handout #18 The sequence of this chain is The read as AGT. read End Handout #18 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 28 C. Double Helix Model: Watson and Crick Watson and Crick, 1953. Handout #19 - outline of the information available to Watson and Crick Handout at the time they formulated their model in 1953. at Two sets of data: Two Erwin Chargaff: Chemical composition of DNA Rosalind Franklin and Maurice Wilkins: X-ray Diffraction of DNA Rosalind 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 29 Erwin Chargaff's Rules 1. [pyrimidines] = [purines]; [T + C] = [A + G] 2. [T] = [A]; [C] = [G] 3. A + T varies among different organisms. G+C 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 30 A + T/G + C Ratio in DNA from various organisms 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 31 Rosalind Franklin: discoverer of DNA crystal structure 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 32 Rosalind Franklin’s X-ray Diffraction Pattern of DNA 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 33 X-Ray Diffraction Data of Rosalind Franklin and Maurice X-Ray Wilkens Wilkens 1. DNA is helical. 2. Two regular patterns of 3.4 nm and 0.34 nm. 3. Consistent radius. 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 34 James Watson and Francis Crick with their DNA model 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 35 Watson and Crick, Nature 1953 Original model for the structure of the DNA Proposed that DNA was a “double helix:” -Sugar-phosphate backbone shown as a ribbon. -Bases as horizontal lines. 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 36 Structure of DNA …phosphate diester groups joining ß-Dphosphate deoxyribofuranose residues with deoxyribofuranose 3',5' linkages. …. the two chains run in opposite the directions. directions …The bases are on the inside of the helix and the phosphates on the outside… …a single base from one chain being hydrogen - bonded to a single base from the other chain base …one of the pair must be a purine and the other a pyrimidine for bonding to occur. 2/16/10 It has not escaped our notice that the specific pairing we have postulated immediately suggests postulated a possible copying mechanism for possible the genetic material. the 37 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression Milestones of Modern Genetics April 25, 2003 — 50th anniversary of the April discovery of DNA 2/16/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 38 Main Features of the Watson and Crick Double Helix Model Main for DNA for 1. Two polynucleotide chains form a right-handed double Two helix. helix. 2. Helix diameter = 2 nm. 3. Two polynucleotide chains are anti-parallel. 4. Sugar phosphate backbone (negative charge) is oriented Sugar to the outside; the bases face the interior of the molecule. The bases are planar and "stack" on one another. another. 5. Opposite strands are bonded by hydrogen bonds. 6. Bases are 0.34 nm apart. One full turn of the helix is 3.4 Bases nm. There are 10 bases/turn (1Å = 0.1 nm). nm. 7. There is a major and a minor groove between the There backbone. backbone. 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 39 The structure of DNA 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 40 Molecular Structure of DNA Molecular Structure of DNA a) 3-d models b) ribbon model with base pairing c) major/minor groove 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 41 Models of DNA 20Å Major Major Groove Groove 3.4Å Stick Model Space Filling Model Minor Minor Groove Groove End End View View Rose Window St. John The St. Divine NYC Divine BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 42 DNA ball-and-stick 3-D model 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 43 Two representations of the DNA double helix 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 44 Complementary base pairing Handout #20 - illustrates the two base pairs of DNA Handout two Complementary base pairing: always have A opposite T, C opposite G. Why? 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 45 Base pairing in DNA 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 46 Complementary base pairing Handout #20 - illustrates the two base pairs of DNA Handout two Complementary base pairing: always have A opposite T, C opposite G. Why? Polarity of chains: The two polynucleotide chains are “antiparallel”. The See Figure 7-8 in text. See 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 47 Antiparallel strands Griffiths et al. Text book: Griffiths Figure 7-8b: Good illustration to show that the two strands Figure are “antiparallel”. So base pairing occurs in an antiparallel manner. So Also shows bonding between bases, but the Also hydrogens are not shown between the chains. You should be able to recognize the 3’ and 5’ ends: -3’ end has OH group. -3’ -5’ end has a phosphate group. Refer to Handout #18. The 3’ end has a hydroxyl group: Refer this is important for DNA synthesis. this 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 48 DNA Structure ­ 3’ 5’ P O C O N H N H H N N O N O CH H N N H N O H H N N H C H N 2/16/10 3’ O BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression O P O - H C H C G O O N H O P O P O H N N O N O O H C H A T O ­ DNA is also a polyanion with a DNA negative charge on every H phosphodiester bond. Electrostatic repulsion causes DNA to writhe or twist violently in solution. Without stabilizing forces, phospho-diester bonds - O will break and the DNA will disintegrate. - 5’ 49 O P O -O O DNA is a plectonemic coil DNA consisting of two poly-nucleotide chains of DNA. Because the 1' carbon of the sugar bonded to the nitrogenous base limits the rotation of the base, the strands of DNA must be arranged in an antiparallel fashion. antiparallel O O - Midterm 2 Exam Cut Off here at slide 49. BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression What are the factors contributing to DNA stability? ­ Ionic bonds ­ Hydrophobic interactions π -bonds ­ Van der Waals interaction ­ Hydrogen bonds ­ 2/16/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 51 Factors contributing to DNA stability ­ 1. Ionic bonds involve an attraction between unlike charges (e.g., 1. Ionic between the (-) carboxyl group of aspartic acid and the (+) amino group of lysine). The ability of cations like Na+ to neutralize the effects of of electrostatic repulsion is a good example of the stabilizing effects of electrostatic ionic bonds. Na+ acts as a counter ion that neutralize the (-) charge on ionic counter the phosphodiester bonds. Writhing subsides and the backbone of the Writhing helix becomes more rigid or stiff. This promotes the stacking of the stacking bases, one over the other. All of this leads to greater stability of the helix. This phenomenon underscores the reasons sodium is typically added to DNA solutions in the laboratory. DNA + Na Cl + H2O = Na+ Cl- O2/16/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 52 DNA stability: Ionic bonds ­ ­ The ability of cations like Na+ to neutralize the effects of electrostatic repulsion is a good example of the stabilizing effects of ionic bonds. Na+ repulsion acts as a counter ion that binds to the (-) charge on the phosphate counter group. Writhing subsides and the backbone of the helix becomes more rigid or Writhing stiff. This promotes the stacking of the bases, one over the other. All of stacking this leads to greater stability of the helix. This phenomenon underscores the reasons NaCl is typically added to DNA solutions in the laboratory. the - -- - Na -- + --- - - ------ 2/16/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 53 DNA stability: Hydrophobicity ­ Hydrophobicity stabilizes DNA by forming ordered shells of water around each ordered base. This ordering of water molecules into shells decreases entropy (or the decreases tendency toward molecular randomness) and therefore is thermodynamically unfavorable. For this reason, shells tend to fuse together, thus reducing order or shells increasing entropy. Since fewer water molecules are ordered in fused shells as compared to shells around individual bases, a smaller number of water molecules remain in an ordered state. This increased entropy is thermodynamically favorable. favorable. = H20 2/16/10 BIS101001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 54 Factors contributing to DNA stability ­ 3. π -Bonds. The nitrogenous bases in DNA tend to stack on top of each 3. The other. This is due to π -bonds between nitrogens in adjacent bases. A π -bonds bond is formed when electrons in two, 2p-orbitals pair and overlap side by bond side. The tendency for one pair of bases to stack is dependent on whether or not the adjacent pair of bases is stacked. In other words, stacking is cooperative because stacking promotes stacking. cooperative N2p N N2s N2p N 2/16/10 x π y y z α N2s x z 55 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression Base Stacking ­ Two views of base stacking in DNA. Figure 1 shows a van der Waal Two space filling model of single stranded DNA. The DNA backbone is shown as a yellow ribbon. On the right (Fig. 2), hydrogen bonding between between complementary base pairs and between stacked bases are shown (black arrow). shown BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 56 2/16/10 DNA stability:van der Waals interactions ­ van der Waals forces also contribute to DNA structure. Because of van fluctuating charge distributions in atoms and molecules (dipole moments), they will attract each until they reach a point where the attractive force and the repulsive force balance out. This occurs when outer shell electrons in each atom begin to overlap. Half the distance between their respective nuclei is called Van der Waals radii. These forces are about Van These as strong as the weakest H bonds and therefore promote DNA stability. These forces are particularly important in DNA/protein interactions where a close fit between molecules is required. close 2/16/10 s orbitals of Cl2 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 57 DNA stability: Hydrogen bonds ­ Hydrogen bonds are weak; only -0.3 to -3.0 kcal/mol (Gibbs energy) above thermal noise. Hydrogen bonds are only second to the phosphodiester bonds in terms of contribution to DNA stability. The weakest hydrogen bond in DNA is -0.9 kcal/mol (AA), whereas thermal noise is about +0.6 kcal/mol. kcal/mol Watson/Crick base pairing between antiparallel strands of DNA: Watson/Crick base q ­ q (a) reduces rotation around the phosphodiester bond and thus reducing writhing and (a) reduces helix instability (b) protects reactive groups on the bases from chemical modification that could lead (b) protects to mutation. The weak nature of these bonds allows strands of DNA to separate or denature during replication or transcription. denature ­ It should also be noted that because of intramolecular complementarity, single It intramolecular single stranded DNA molecules (and RNA molecules) can form double helices with themselves. The transfer RNA (tRNA) is a good example of this. themselves. Gibbs energy (Gibbs free energy) is the thermodynamic potential that measures the “useful” or “processiinitiating” work obtainable in a closed thermodynamic system. As a process changes states (e.g., from nitiating” double strand DNA to single strand DNA) Gibbs energy changes from -∆ G to + ∆ G. double to 2/1/6/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 58 5. DNA forms The Watson and Crick model for the double helix is also The known as: known “B-DNA”. -this form is in the cell. -this -forms right-handed double helix. -forms But there are other forms of DNA also: A DNA: also forms right-handed double helix Z DNA: forms left-handed double helix (zigzag). Space-filling models of DNA: A, B, Z forms 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 59 Alternate DNA Forms: January 9, 1981 2/16/10 BIS101­001, Winter 2010Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 60 Alternate DNA Forms ­ DNA structure can vary in nature. Although the double-stranded B helix is the DNA predominant form, other helical and some completely unexpected forms can be found. be B-DNA A-DNA Z-DNA 2/16/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 61 Alternate DNA Structures: A-form DNA ­ The A form helix is not the predominant form of doublepredominant stranded RNA ­ A-form DNA or RNA has 11 A-form bases per turn instead of the 10 bases in B-DNA the ­ A-form DNA is wider and A-form shorter than the B helix, and the distinction between the major and minor grooves is reduced reduced ­ This helix is favored under This conditions ofIS101­001, Winter 2010—Genes and Gene Expression dehydration conditions BIS101­001, Winter 2010—Genes and Gene Expression 2/16/10 B A-DNA Minor groove Major groove 62 Alternate DNA Structures: Z form DNA ­ ­ ­ ­ ­ The Z helix (so-named because the The helix backbone has a zigzag shape) is a more radical departure from the B helix theme. radical Although double stranded, the Z helix is a left-handed helix and has 12 base pairs per turn instead of the 10 in B-DNA and 11 in A-DNA. Z DNA appears longer and slimmerllooking than the B helix. ooking Unlike B-DNA and A-DNA, Z-DNA is Unlike dependent on the nucleotide sequence. Alternating G and C promotes the formation of Z-DNA. Therefore, stretches of Z-DNA can be found interspersed in Bof DNA chromosomes. DNA The presence of regions of Z DNA near The genes on the same molecule can influence their expression by promoting protein binding. protein 2/16/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 63 D. RNA structure RNA is a polymer like DNA but RNA is: 1. single stranded. 2. has a lot of structure: in solution, RNA forms a right-handed 2. helix, but in the cell, RNA also forms other types of structures due to intramolecular complementarity. to 3. can base pair with complimentary strands of DNA, RNA. 4. RNA can base pair with itself: forms “hairloop” structure by 4. complementary base pairing with itself. complementary 5. has uracil, U = A (U base pairs with A). 6. has ribose with a hydroxyl on 2’ C. This 2’-hydroxyl makes 6. RNA more prone to hydrolysis and less stable than DNA, especially at high temperatures. especially 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 64 Intramolecular complementarity of RNA Intramolecular Transfer RNA Transfer 2/16/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 65 RNA is classified by its type and function Types of RNA: 1. messenger RNA (mRNA) -product of transcription, -RNA copy of DNA, -used by cytoplasm for protein synthesis. 2. ribosomal RNA (rRNA) -structural components of ribosomes, -structural -important for correct translation of proteins -important -rRNA catalyzes formation of a peptide bond during -rRNA protein synthesis. protein 3. transfer RNA (tRNA) -adaptor molecule important in protein synthesis. 4. small RNA (sRNA) -a whole class -function in RNA processing 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 66 Lecture summary: Topic X. Genetic Material- DNA and RNA A. DNA as genetic material 1. Griffith experiment 2. Avery, MacLeod, and McCarty experiment 3. Hershey and Chase experiment B. Chemical composition of DNA/RNA 1. Nucleotides (Handout #17) 2. Phosphodiester bond (Handout #18) C. Double Helix Model 1. X-ray diffraction (Franklin and Wilkens) (Handout #19) 2. Chargaff's rules ((Handout #19) 3. Base pairing (Handout #20) 4. Watson and Crick model (Handout #19) D. RNA structure 03/29/10 BIS101­001, Winter 2010—Genes and Gene Expression BIS101­001, Winter 2010—Genes and Gene Expression 67 ...
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This note was uploaded on 03/29/2010 for the course BIO SCI 101 taught by Professor Kimsey during the Spring '10 term at UC Davis.

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