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Chapter 11 PowerPoint PPT Presentation

Chapter 11. Structure of Nucleic Acids Biochemistry by Reginald Garrett and Charles Grisham. Essential Question. What is the higher-order structure of DNA and RNA, and what methodologies have allowed scientists to probe these structures and the functions that derive from them?. Outline.

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Chapter 11 l.jpg

Chapter 11

Structure of Nucleic Acids



Reginald Garrett and Charles Grisham

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Essential Question

  • What is the higher-order structure of DNA and RNA, and what methodologies have allowed scientists to probe these structures and the functions that derive from them?

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  • How Do Scientists Determine the Primary Structure of Nucleic Acids?

  • What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt?

  • Can the Secondary Structure of DNA Be Denatured and Renatured?

  • What is the Tertiary Structure of DNA?

  • What Is the Structure of Eukaryotic Chromosomes?

  • Can Nucleic Acids Be Chemically Synthesized?

  • What Is the Secondary and Tertiary Structure of RNA?

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11.1- How Do Scientist Determine the Primary Structure of Nucleic Acids?

Sequencing Nucleic Acids

  • Chain termination method (dideoxy method), developed by F. Sanger.

  • Base-specific chemical cleavage, developed by Maxam and Gilbert.

  • Both use autoradiography - X-ray film develops in response to presence of radioactive isotopes in nucleic acid molecules.

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DNA Replication

  • DNA is a double-helical molecule.

  • Each strand of the helix must be copied in complementary fashion by DNA polymerase.

  • Each strand is a template for copying.

  • DNA polymerase requires template and primer.

  • Primer: an oligonucleotide that pairs with the end of the template molecule to form dsDNA.

  • DNA polymerases add nucleotides in 5'-3' direction.

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Chain Termination Method

Based on DNA polymerase reaction

  • Run four separate reactions.

  • Each reaction mixture contains dATP, dGTP, dCTP and dTTP, one of which is P-32-labelled.

  • Each reaction also contains a small amount of one dideoxynucleotide: either ddATP, ddGTP, ddCTP or ddTTP.

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Chain Termination Method

  • Most of the time, the polymerase uses normal nucleotides and DNA molecules grow normally.

  • Occasionally, the polymerase uses a dideoxynucleotide, which adds to the chain and then prevents further growth in that molecule.

  • Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide.

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Chain Termination Method

  • Run each reaction mixture on electrophoresis gel.

  • Short fragments go to bottom, long fragments on top.

  • Read the "sequence" from bottom of gel to top

  • Convert this "sequence" to the complementary sequence.

  • Now read from the other end and you have the sequence you wanted - read 5' to 3.

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Base-Specific Chemical Cleavage Method

  • By Maxam and Gilbert

  • G: Me2SO4 at pH 7

  • A + G: Me2SO4 at pH 2

  • C + T: NH2NH2

  • C: NH2NH2 (1 or 2M NaCl)

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11.2 – What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt?

Secondary Structure

  • See Figure 11.7 for details of DNA secondary structure

  • Sugar-phosphate backbone outside

  • Bases (hydrogen-bonded) inside

  • Right-twist closes the gaps between base pairs to 3.4 A (0.34 nm) in B-DNA

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The “canonical” base pairs

See Figure 11.7

  • The canonical A:T and G:C base pairs have nearly identical overall dimensions.

  • A and T share two H bonds.

  • G and C share three H bonds.

  • G:C-rich regions of DNA are more stable.

  • Polar atoms in the sugar-phosphate backbone also form H bonds.

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The DNA double helix is a stable structure.

Factors for stablizing the DNA helix structure:

  • H-bonds: from AT & GC pairs &

    from polar atoms in the sugar-phosphate backbone with surrounding water.

  • Electrostatic interactions: The negatively charged phosphate groups are on the outside so that the repulsive effects are minimized. Mg+2 ions bind strongly to them.

  • Van der Waals and hydrophobic interactions:

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Major and minor grooves

See Figures 11.7, 11.8

  • The "tops" of the bases (as we draw them) line the "floor" of the major groove.

  • The major groove is large enough to accommodate an -helix from a protein.

  • Regulatory proteins (transcription factors) can recognize the pattern of bases and H bonding possibilities in the major groove.

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Figure 11.9Helical twist and propeller twist in DNA.(a) Successive base pairs in B-DNA show a rotation with respect to each other (so-called helical twist) of 36° or so, as viewed down the cylindrical axis of the DNA. (b) Rotation in a different dimension—propellertwist—allows the hydrophobic surfaces of bases to overlap better. The view here is edge-on to two successive bases in one DNA strand (as if the two bases on the right-hand strand of DNA in (a) were viewed from the right-hand margin of the page; dots represent end-on views down the glycosidic bonds). Clockwise rotation (as shown here) has a positive sign. (c) The two bases on the left-hand strand of DNA in (a) also show positive propeller twist (a clockwise rotation of the two bases in (a) as viewed from the left-hand margin of the paper).

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Comparison of A, B, Z DNA

See Table 11.1

  • A: right-handed, short and broad, 2.3 Å, 11 bp per turn

  • B: right-handed, longer, thinner, 3.32 Å, 10 bp per turn

  • Z: left-handed, longest, thinnest, 3.8 Å, 12 bp per turn

  • See Figure 11.10

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Discovered by Alex Rich

  • Found in G:C-rich regions of DNA

  • The alternating pyrimidine–purine sequence of Z-DNA is the key to the “left-handedness” of this helix.

  • G goes to syn conformation

  • C stays anti but whole C nucleoside (base and sugar) flips 180 degrees

  • Result is that G:C H bonds can be preserved in the transition from B-form to Z-form!

  • 5-Methylcytosine in Z-DNA is related to gene regulation.

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  • The double helix is a very dynamic structure.

  • - flexible. It can rotate bonds forming spherical shape, not random coil.

  • - Influenced by base sequence, which can be recognized by regulatory proteins to activate or suppress gene expression.

  • Intercalating agents distort the double helix.

  • - Aromatic macrocycles, flat hydrophobic molecules insert between the stacked base pairs, causing unwinding of the backbone to be more ladderlike.

  • Dynamic nature of the DNA double helix in solution --- metastable alternatives to standard B-conformation.

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11.3 – Can the Secondary Structure of DNA Be Denatured and Renatured?

See Figure 11.17

  • When DNA is heated to 80+C, its UV absorbance increases by 30-40%.

  • This hyperchromic shift reflects the unwindingof the DNA double helix.

  • Stacked base pairs in native DNA absorb less light.

  • When T is lowered, the absorbance drops, reflecting the re-establishment of stacking.

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Figure 11.17These c0t curves show the rates of reassociation of denatured DNA from various sources and illustrate how the rate of reassociation is inversely proportional to genome complexity. The DNA sources are as follows: poly A+poly U, a synthetic DNA duplex of poly A and poly U polynucleotide chains; mouse satellite DNA, a fraction of mouse DNA in which the same sequence is repeated many thousands of times; MS-2 dsRNA, the double-stranded form of RNA found during replication of MS-2, a simple bacteriophage;

C0: concentration of completely denatured DNA at t= 0

t1/2: the time for half of the DNA to renature

C0t1/2 = 1/k2

T4 DNA, theDNA of a more complex bacteriophage; E. coli DNA, bacterial DNA; calf DNA (nonrepetitive fraction), mammalian DNA (calf) from which the highly repetitive DNA fraction (satellite DNA) has been removed. Arrows indicate the genome size (in bp) of the various DNAs.

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Nucleic acid hybridization: Different DNA strands of similar sequence can form hybrid duplexes

FIGURE 11.18 Solutions of human DNA (red) and mouse DNA (blue) are mixed and denatured, and the single strands are allowed to reanneal. About 25% of the human DNA strands form hybrid duplexes (one red and one blue strand) with mouse DNA.

Fig. 11-18, p.352

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Density gradient centrifugation is a common method of separating macromolecules, particularly nucleic acids, in solution. A cell extract is mixed with a solution of CsCl to a final density of about 1.7 g/cm3 and centrifuged at high speed (40,000 rpm, giving relative centrifugal forces of about 200,000 g). The biological macromolecules in the extract will move to equilibrium positions in the CsCl gradient that reflect their buoyant densities.

Fig. 11A-1, p.374

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11.4 – What Is the Tertiary Structure of DNA?

  • Linear: Chromosomes of eukaryotes

  • Circular: Bacterial chromosomes, plasmids, some viruses

  • In duplex DNA, ten bp per turn of helix.

  • Circular DNA sometimes has more or less than 10 bp per turn - a supercoiled state.

  • Enzymes called topoisomerases or gyrases can introduce or remove supercoils.

  • Cruciforms occur in palindromic regions of DNA.

  • Negative supercoiling may promote cruciforms.

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11.5- What Is the Structure of Eukaryotic Chromosomes?

  • Human DNA’s total length is ~2 meters!

  • This must be packaged into a nucleus that is about 5 m in diameter.

  • This represents a compression of more than 100,000!

  • It is made possible by wrapping the DNA around protein spools called nucleosomes and then packing these in helical filaments.

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Nucleosome Structure

  • Chromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteins.

  • Histone octamer structure has been solved.

  • Nonhistone proteins are regulators of gene expression.

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Telomeres and Tumors

Telomerase, a RNA-containing DNA polymerase, maintain the integrity of chromosome against degradation. Most normal somatic cells lack telomerase. They lose 50 nucleotides per cell cycle and eventually lead to chromosome instability and cell death.

10001700 repeats in human germline cells

20 different types of cancer cells were found to contain telomerase activity.

Telomerase RNA serves as the template for the DNA polymerase activity.


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11.6 – Can Nucleic Acids Be Chemically Synthesized?

  • Laboratory synthesis of nucleic acids requires complex strategies.

  • Functional groups on the monomeric units are reactive and must be blocked.

  • Correct phosphodiester linkages must be made.

  • Recovery at each step must high!

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Solid Phase Oligonucleotide Synthesis

  • Dimethoxytrityl group blocks the 5'-OH of the first nucleoside while it is linked to a solid support by the 3'-OH.

  • Step 1: Detritylation by trichloroacetic acid exposes the 5'-OH.

  • Step 2: In coupling reaction, second base is added as a nucleoside phosphoramidate.

  • Step 3: capping with acetic anhydride blocks unreacted 5’-OHs of N-1 from further reaction.

  • Step 4: Phosphite linkage between N-1 and N-2 is reactive and is oxidized by aqueous iodine (I2) to form the desired, and more stable, phosphate group.

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11.7 – What Is the Secondary and Tertiary Structure of RNA?

Transfer RNA

  • Extensive H-bonding creates four double helical domains, three capped by loops, one by a stem.

  • Only one tRNA structure (alone) is known.

  • Phenylalanine tRNA is "L-shaped“.

  • Many non-canonical base pairs found in tRNA.

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Ribosomal RNA

Ribosomes synthesize proteins

  • All ribosomes contain large and small subunits.

  • rRNA molecules make up about 2/3 of ribosome.

  • High intrastrand sequence complementarity leads to extensive base-pairing.

  • Secondary structure features seem to be conserved, whereas sequence is not.

  • There must be common designs and functions that must be conserved.

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