Chapter 11 Structure of Nucleic Acids

Transcription

1 Reginald H. Garrett Charles M. Grisham Chapter 11 Structure of Nucleic Acids 11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids? Two simple tools have made nucleic acid sequencing easier than polypeptide sequencing: The type II restriction endonucleases that cleave DNA at specific oligonucleotide sites Gel electrophoresis, which is capable of separating nucleic acid fragments that differ from one another in length by just a single nucleotide

2 11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids? Chain termination method (dideoxy method), developed by Frederick Sanger is the basis for nearly all DNA sequencing today The method takes advantage of the DNA polymerase reaction, which copies a DNA strand in complementary fashion to form a new second strand 11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids? Figure 11.1 DNA replication yields two daughter DNA duplexes identical to the parental DNA molecule.

3 11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids? Figure 11.2 Primed synthesis of a DNA template by DNA polymerase, using the four deoxynucleoside triphosphates as the substrates How Do Scientists Determine the Primary Structure of Nucleic Acids? 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 template molecule to form dsdna DNA polymerases add nucleotides in 5 3' direction

4 Chain Termination Method A template DNA with a complementary primer is copied by DNA polymerase in the presence of datp, dctp, dgtp, dttp Solution contains small amounts of the four dideoxynucleotide analogs of these substrates, each of which contains a distinctive fluorescent tag, illustrated here as: Orange for ddatp Blue for ddctp Green for ddgtp Red for ddttp Occasional incorporation of a dideoxynucleotide terminates further synthesis of that strand Figure 11.3 The chain termination method of DNA sequencing.

5 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

6 Chain Termination Method Reaction mixtures can be separated by capillary electrophoresis 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' The set of terminated strands can be separated by capillary electrophoresis

7 High-Throughput DNA Sequencing by the Light of Fireflies The importance of DNA sequence information has motivated development of more rapid and efficient DNA sequencing technologies 454 Technology relies on DNA polymerase but does not involve chain termination Multiple copies of template DNA molecules are immobilized on microscopic beads Reagents for primed synthesis are passed over the beads in sequential order (one at a time! TAGC) Pyrophosphate release is monitored by light emission via ATP sulfurylase and luciferase reactions High-Throughput DNA Sequencing by the Light of Fireflies DNA polymerase produces PP i ATP sulfurylase: PP i + APS ATP + SO 4 2- Luciferase: ATP + luciferin + O 2 AMP + PP i + CO 2 + oxyluciferin + light Structures of luciferin and oxyluciferin. Light detection confirms that addition of a dnmp by primed synthesis has occurred.

8 High-Throughput DNA Sequencing by the Light of Fireflies 11.2 What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt? Polynucleotide strands are flexible Each deoxyribose-phosphate segment of the backbone has six degrees of freedom (Fig 11.4a) Furanose rings are not planar but instead adopt puckered ( 皺摺的 ) conformations, four of which are shown in Figure 11.4b A seventh degree of freedom per nucleotide arises because of free rotation about the C1'-N glycosidic bond This freedom allows the plane of the base to rotate relative to the path of the polynucleotide strand

9 11.2 What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt? Figure 11.4 The six degrees of freedom in the deoxyribose-po 4 units of the polynucleotide chain What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt? Figure 11.4 Four puckered conformations of the furanose rings.

10 11.2 What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt? Figure 11.4 Free rotation about the C1'-N glycosidic bond What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt? The stability of the DNA double helix is due to: Hydrogen bonds between base pairs Electrostatic interactions mutual repulsion of phosphate groups, which makes them most stable on the helix exterior Van der Waals and hydrophobic interactions base pair stacking interactions

11 The Watson-Crick base pairs See Figure 11.6 The A:T pair and G:C pair have nearly identical 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 The Watson-Crick base pairs The A:T pair and G:C pair have nearly identical dimensions

12 The Watson-Crick base pairs Figure 11.6 Watson- Crick A:T and G:C base pairs. All H-bonds in both base pairs are straight. The Watson-Crick base pairs Figure 11.6 Watson- Crick A:T and G:C base pairs. All H- bonds in both base pairs are straight.

13 Major and minor grooves See Figures 11.6, 11.7 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 alpha helix from a protein Regulatory proteins (transcription factors) can recognize the pattern of bases and the H bonding possibilities in the major groove Major and minor grooves Figure 11.7 The major and minor grooves of B-DNA.

14 Double Helical Structures Can Adopt a Number of Stable Conformations The DNA double helix can adopt several stable conformations Helical twist is the rotation of one base pair relative to the next, around the axis of the double helix Successive base pairs in B-DNA show a mean rotation of 36 with respect to each other Propellor twist involves rotation around a different axis, namely an axis perpendicular to the helix axis See Figure 11.8 Double Helical Structures Can Adopt a Number of Stable Conformations 36 o Two base pairs with 36 o of right-handed twist Figure 11.8 Helical twist and propellor twist in DNA (a) Successive base pairs in B-DNA show a rotation with respect to each other.

15 Double Helical Structures Can Adopt a Number of Stable Conformations Figure 11.8 Helical twist and propellor twist in DNA. (b) Rotation in a different dimension propellor twist allows the hydrophobic surfaces of bases to overlap better Double Helical Structures Can Adopt a Number of Stable Conformations Helical twist and propellor twist in DNA. (c) Each of the bases in a base pair shows positive propellor twist as viewed along the N-glycosidic bond. Note how the hydrogen bonds between bases are distorted by this motion, yet remain intact.

16 Double Helical Structures Can Adopt a Number of Stable Conformations Figure 11.9a The B-form of the DNA double helix. In B-form, the pitch (the distance required to complete one helical turn) is 3.4 nm (10 to 10.6 bp ). 12 bp Double Helical Structures Can Adopt a Number of Stable Conformations Figure 11.9b The A-form of the DNA double helix. The pitch of the A-form helix is 2.46 nm (11 bp); thus the A- form is a shorter, wider structure than the B-form. 12 bp

17 Z-DNA is a Conformational Variation in the Form of a Left-Handed Double Helix Figure 11.9c The Z-form of double helical DNA. G:C-rich regions The N-glycosyl bonds of G residues are rotated 180 with respect to their conformation in B-DNA, so now the purine ring is in the syn rather than the anti conformation. 6 bp Z-DNA is a Conformational Variation in the Form of a Left-Handed Double Helix Figure 11.9c The Z-form of double helical DNA. The C residues remain in the anti form. Because the G ring is flipped, the C ring must also flip to maintain normal Watson-Crick base pairing. G:C H bonds can be preserved in the transition from B-form to Z-form!

18 Comparison of B and Z DNA Figure Comparison of the deoxyguanosine conformation in B- and Z-DNA. The Change in Topological Relationships of Base Pairs from B- to Z-DNA Figure It is topologically possible for G to go syn and the C nucleoside to undergo rotation by 180 without breaking and re-forming the G:C hydrogen bonds.

19 Comparison of A, B, Z DNA Intercalating Agents Distort the Double Helix The double helix is a very dynamic structure Because it is flexible, aromatic macrocycles flat hydrophobic molecules composed of fused, heterocyclic rings, can slip between the stacked pairs of bases The bases are force apart to accommodate these intercalating agents Ethidium bromide Acridine orange Actinomycin D

20 Intercalating Agents Distort the Double Helix Figure The structures of ethidium bromide, acridine orange, and actinomycin D, three intercalating agents, and their effects on DNA structure. Alternative H-Bonding Interactions Give Rise to Novel DNA Structures Cruciform ( 十字形 )structures arise from inverted repeats. In such structures, the normal interstrand base pairing is replaced by intrastrand pairing. Figure Self-complementary inverted repeats can rearrange to form H-bonded cruciform stem-loop structures. Cruciforms are not as stable as normal DNA, because an unpaired segment must exist in the loop.

21 Hoogsteen Base Pairs and DNA Multiplexes Karst Hoogsteen found some A:T and G:C base pairs that are different from the canonical structures. In both A:T and G:C Hoogsteen base pairs, the purine N-7 atom is an H-bond acceptor. Figure Hoogsteen base pairs: A:T (left) and C+:G (right). Hoogsteen Base Pairs and DNA Multiplexes Figure Base triplets can form when a purine interacts with one pyrimidine by Hoogsteen base pairing and another by Watson-Crick base pairing.

22 H-DNA is Triplex DNA Made of One Purine-Rich Strand and Two Pyrimidine-Rich Strands Figure H-DNA. The Hoogasteen base-paired pyrimidine-rich strand in triplex structure is yellow. DNA Quadruplex Structures G-quadruplexes are cyclic arrays of four G residues united through Hoogsteen base pairing. Figure 11.17a G-quadruplex showing the cyclic array of guanines linked by Hoogsteen hydrogen bonds.

23 DNA Quadruplex Structures Figure 11.17b Four G-rich polynucleotide strands in parallel alignment with all bases in anti conformation. DNA Quadruplex Structures Figure 11.17c Antiparallel dimeric hairpin quadruplex formed form (dg 4 T 4 G 4 ) 2

24 11.3 Can the Secondary Structure of DNA Be Denatured and Renatured? When DNA is heated to 80+ degrees Celsius, its UV absorbance increases by 30-40% This hyperchromic shift reflects the unwinding of 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 The lower the ionic strength, the lower the melting temperature. ph extremes (>10 or < 2.3) and storng H- bonding solutes (e.g. urea) also denature DNA duplexes Can the Secondary Structure of DNA Be Denatured and Renatured? Figure Heat denaturation of DNA from various sources. The higher the GC content the higher its melting temp.

25 The Buoyant Density of DNA Density gradient ultracentrifugation is a useful way to separate and purify nucleic acids. The net movement of solute particles in an ultracentrifuge is the result of two processes: diffusion (from regions of higher concentration to regions of lower concentration) and sedimentation due to centrifugal force. Single-Stranded DNA Can Renature to Form DNA Duplexes Denatured DNA will renature to re-form the duplex structure if the denaturing conditions are removed Renaturation requires reassociation of the DNA strands into a double helix, a process termed reannealing For this to occur, the strands must realign so that their complementary bases are once again in register and the helix can be zippered up

26 Single-Stranded DNA Can Renature to Form DNA Duplexes Figure Steps in the thermal denaturation and renaturation of DNA. Nucleic Acid Hybridization: Different DNA Strands of Similar Sequence Can Form Hybrid Duplexes If DNA from two different species are mixed, denatured, and allowed to cool slowly, hybrid duplexes may form, provided the DNA from one species is similar in sequence to the other The degree of hybridization is a measure of the sequence similarity between the two species 25% of the DNA from a human forms hybrids with mouse DNA, implying some sequence similarity Hybridization is a common procedure in molecular biology for revealing evolutionary relationships and for identifying specific genes

27 Nucleic Acid Hybridization: Different DNA Strands of Similar Sequence Can Form Hybrid Duplexes Figure Solutions of human DNA (red) and mouse DNA (blue) are mixed and denatured, and the single strands are allowed to reanneal. About 25% of human DNA forms hybrid duplexes with mouse DNA Can DNA Adopt Structures of Higher Complexity? In duplex DNA, there are 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

28 Supercoils Are One Kind of Structural Complexity in DNA Double-stranded circular DNA forms supercoils, if the strands are underwound (-), or overwound (+). Two strands are wound about each other once every 10 bp. Figure Toroidal and interwound varieties of supercoiling. Supercoiled DNA is characterized by a Linking Number (L), Twist (T), and Writhe (W) L=T+W Figure Linking number (L) is sum of twist (T) and writhe (W). Twist : the number of helical turns. Writhe: the number of supercoils.

29 Supercoiled DNA is characterized by a Linking Number (L), Twist (T), and Writhe (W) L=T+W -1 - Figure Linking number (L) is sum of twist (T) and writhe (W). Twist : the number of helical turns. Writhe: the number of supercoils. DNA Gyrase is a topoisomerase that introduces negative supercoils into DNA Negative supercoils cause a torsional stress on the molecule, so the molecule tends to unwind. Negative supercoiling makes it easier to separate DNA strands and access the information encoded by the sequence. Figure 11.23a A model for the action of bacterial DNA gyrase (topoisomerase II).

30 DNA Gyrase is a topoisomerase that introduces negative supercoils into DNA Figure 11.23b Conformational changes in the enzyme allow an intact region of the DNA duplex to pass between the cut ends. The cut ends are religated (3), and the covalently complete DNA duplex is released from the enzyme. The circular DNA now contains two negative supercoils (4). Negative Supercoiling has the Potential to Cause Localized Unwinding in DNA Figure A 400-bp circular DNA molecule in different topological states: (a) relaxed, (b) negative supercoils distributed over the entire length, and (c) negative supercoils creating a localized single-stranded region.

31 Negatively Supercoiled DNA Can Arrange into a Toroidal State Figure The toroidal state is stabilized by wrapping around proteins that serve as spools for the DNA ribbon 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 micrometers in diameter This represents a compression factor 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 These filaments are thought to arrange in loops associated with the nuclear matrix

32 Nucleosomes Are the Fundamental Structural Unit in Chromatin Histones and nonhistone chromosomal proteins are the two classes of chromatin proteins. Five distinct histones are known: H1, H2A, H2B, H3, and H4. Pairs of histones H2A, H2B, H3, and H4 aggregate to form octameric core structures; the DNA helix is wound around these core octamers, creating nucleosomes. Nucleosome Structure Chromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteins Histone octamer structure has been solved (without DNA by Moudrianakis, and with DNA by Richmond) Nonhistone proteins are regulators of gene expression

33 The Structure of the Nucleosome a Histone Octamer wrapped with DNA Figure Higher-Order Structural Organization of Chromatin Gives Rise to Chromosomes Figure A model for chromosome structure, human chromsome 4, showing nucleosomes in the beads on a string motif.

34 Higher-Order Structural Organization of Chromatin Gives Rise to Chromosomes 30-nm fiber Figure A model for chromosome structure, human chromsome 4. A higher order of chromatin structure is created when the array of nucleosomes is wound in the fashion of a solenoid. Higher-Order Structural Organization of Chromatin Gives Rise to Chromosomes Figure A model for chromosome structure, human chromsome 4. The 30 nm fiber forms long DNA loops of variable length, each containing on average between 60,000 and 150,000 bp.

35 Higher-Order Structural Organization of Chromatin Gives Rise to Chromosomes Figure A model for chromosome structure, human chromsome 4. Electron microscopic analysis of chromosome 4 suggests that 18 loops are arranged radially about the circumference of a single turn to form a miniband unit of the chromosome. Higher-Order Structural Organization of Chromatin Gives Rise to Chromosomes Figure A model for chromosome structure, human chromsome 4. Approximately a million minibands are arranged along a central axis in each of the chromatids of chromosome 4 that form at mitosis.

36 SMC Proteins Establish Chromosome Organization and Mediate Chromosome Dynamics SMC proteins are nonhistone chromosomal proteins SMC proteins form a large superfamily of ATPase involved in higher-order chromosome organization and dynamics. DNA-binding and ATPase abilities Figure SMC protein architecture and function. SMC proteins range from 115 to 165 kd in size. SMC Proteins Set Chromosome Organization and Mediate Chromosome Dynamics Figure SMC protein architecture and function. Shown here is the condensation of DNA into a coiled arrangement through SMC2/SMC4-mediated interactions.

37 Telomeres and Tumors The ends of chromosomes have specialized structures known as telomeres - short, tandemly repeated nucleotide sequences at the ends of the chromosomal DNA Telomeres are added to the ends of chromosomal DNA by an RNA-containing enzyme known as telomerase Most normal somatic cells lack telomerase; thus with every cycle of cell division about 50 nucleotides are lost from each telomere The telomere theory of aging argues that telomere shortening is a factor in cell, tissue and organism aging. Tumor cells have telomerase immortal Telomeres and Tumors Telomeres on human chromosomes.

38 11.6 Can Nucleic Acids Be Synthesized Chemically? 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! Solid-phase methods are used to satisfy some of these constraints DNA synthesizer: synthesis direction 3 5 Limitation: ~150 bases Genes Can Be Synthesized Chemically Theses genes synthesis involves joining a series of synthesized oligonucleotides

39 11.7 What Is the Secondary and Tertiary Structure of RNA? The double-stranded structure of DNA imposes great constraints on its conformational possibilities RNA molecules are typically single-stranded and thus have six to seven degrees of freedom per nucleotide unit Thus RNA molecules have a much greater number of conformational possibilities Complementary sequences in RNA can join via intrastrand base pairing When the base pairing is not complete, a variety of bulges and loops can form, including hairpin stemloop structures 11.7 What Is the Secondary and Tertiary Structure of RNA? Figure Bulges and loops formed in RNA when aligned sequences are not fully complementary.

40 11.7 What Is the Secondary and Tertiary Structure of RNA? A number of defined structural motifs recur within the loops of stem-loop structures, such as U- turns (a loop motif of consensus sequence UNRN, where N is any nucleotide and R is purine), tetraloops (a type of four-base hairpin loop motifs), and bulges (or internal loops) Regions where several stem-loop structures meet are termed junctions Stems, loops, bulges, and junctions are the four basic secondary structural elements in RNA 11.7 What Is the Secondary and Tertiary Structure of RNA? Other tertiary structural motifs arise from coaxial stacking (Figure 11.31), pseudoknot formation (Figure 11.32), and ribose zippers The ribose zipper is a tertiary interaction formed by consecutive hydrogen-bonding between the backbone ribose 2 -hydroxyls from two regions of the chain interacting in an anti-parallel manner.

41 11.7 What Is the Secondary and Tertiary Structure of RNA? Figure Junctions and coaxial stacking in RNA What Is the Secondary and Tertiary Structure of RNA? Figure RNA pseudoknots are formed when a single-stranded region of RNA folds to basepair with a hairpin loop.

42 ribose zipper Transfer RNA Adopts Higher-Order Structure Through Intrastrand Base Pairing In trna, with nucleotides in a single chain, a majority of the bases are hydrogen bonded to one another Hairpin turns bring complementary stretches of bases into contact Extensive H-bonding creates four double helical domains: the acceptor stem, the D loop, the anticodon loop, and the TΨC loop. Phenylalanine trna is "L-shaped" Many non-canonical base pairs found in trna

43 Transfer RNA Adopts Higher-Order Structure Through Intrastrand Base Pairing Figure A general diagram for the structure of trna. Aminoacyl group Transfer RNA Adopts Higher-Order Structure Through Intrastrand Base Pairing Figure Tertiary interactions in yeast phenylalanine trna. Solid lines connect bases that are hydrogen bonded when this cloverleaf pattern is folded into the characteristic trna tertiary structure (see Figure 11.35).

44 trna Tertiary Structure Arises From Interloop Base Pairing Figure trna Tertiary Structure Arises From Interloop Base Pairing Figure The threedimensional structure of yeast phenylalanine trna. The anticodon loop is at the bottom and the acceptor end is at the top right.

45 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 Ribosomal RNA also Adopts Higher-Order Structure Through Intrastrand Base Pairing These secondary structures of several 16S rrnas are based on computer alignment of rrna nucleotide sequences into optimal H-bonding segments. Figure Comparison of secondary structures of 16S-like rrnas from several organisms.

46 rrna Tertiary Structure Figure Detailed structures of ribosomes have been revealed by X-ray crystallography and cryoelectron microscopy. These images reveal details of both tertiary and quaternary interactions that occur when ribosomal proteins combine with rrnas to form the complete ribosome. Riboswitches Act as Regulators of Gene Expression Riboswitches, naturally occurring aptamers, are conserved regions of mrnas that reversibly bind specific metabolites and coenzymes and act as gene expression regulators. Aptamers are oligonucleotide or peptide molecules that bind a specific target molecule.