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Chapter 2: The Structure of DNA
Imagine yourself inside one of those cooling DNA solutions, observing the rebirth of beautifully undulating, semirigid, double-helical threads from the jumble of billions of intertwisted single strands. It is a mind reeling spectacle. Christian de Duve, A Guided Tour of the Living Cell (1984), p. 292
Objectives Describe the structure of DNA Keep its function within living cells in mind
2.2 Primary structure: the components of nucleic acids
Five-carbon sugars Nitrogenous bases The phosphate functional group Components of nucleotides
Edwin Chargaff’s “rules” [A] = [T] [G] = [C] [A] + [G] = [T] + [C] %G+C differs among species but is constant in all cells of an organism within a species. Varies from 22 to 73%
Nucleosides and nucleotides DNA and RNA chains are formed through a series of three steps: 1.A base attached to a sugar is a nucleoside. 2.A nucleoside with one or more phosphates attached is a nucleotide. 3.Nucleotides are linked by 5′ to 3′ phosphodiester bonds between adjacent nucleotides to form a DNA or RNA chain.
The components of a DNA or RNA chain are joined by covalent bonds. A covalent bond is a strong chemical bond formed when electrons are shared between two atoms. These bonds are very stable and do not break spontaneously within cells.
Nomenclature of nucleotides Example: the base cytosine (C) DNA deoxycytidine 5′-triphosphate (dCTP) RNA cytidine 5′-triphosphate (CTP) Generic deoxynucleoside 5′-triphosphate (dNTP) nucleoside 5′-triphosphate (NTP)
The length of RNA and DNA RNA The number of nucleotides (nt) or bases is used as a measure of length. Double-stranded DNA The number of base pairs (bp) is used as a measure of length bp = 1 kilobase pair (kb or kbp) 1,000,000 = 1 megabase pair (Mb or Mbp)
Natural RNAs come in sizes ranging from less than one hundred to many thousands of nucleotides DNA can be as long as several kb to thousands of Mb Oligonucleotides are short chains of single-stranded DNA (< 50 bases)
Significance of 5′ and 3′ The 5′-PO 4 and 3′-OH ends of a DNA or RNA chain are distinct and have different properties By convention, a DNA sequence is written with the 5′ end to the left and the 3′ end to the right
2.3 Secondary structure of DNA
Hydrogen bonds form between the bases Two common “ Watson-Crick” or “complementary” base pairs: Adenine (A) is joined to thymine (T) by two hydrogen bonds. Guanine (G) is joined to cytosine (C) by three hydrogen bonds.
Why aren’t there other stable base pairs present in DNA? May not be able to form two or more hydrogen bonds. Pairing of G with T produces a pair with a similar shape to Watson-Crick base pairs. Fidelity of DNA replication: proofreading and DNA repair mechanisms correct mistakes. GU base-pairing is of importance in RNA structure.
Base stacking provides chemical stability to the DNA double helix The hydrophobic nitrogenous bases stack onto each other without a gap by means of a helical twist. A double-stranded DNA molecule has a hydrophobic core composed of stacked bases.
Hydrophobic bonding is an example of weak van der Waals interactions. A large number of weak van der Waals interactions can significantly increase the stability of a structure, such as the DNA double helix.
Structure of the Watson-Crick DNA double helix Polarity in each strand: 5′ 3′ Two strands are antiparallel Major and minor grooves
The major groove carries a “message” that can be read by DNA binding proteins.. In the major groove, the pattern of hydrogen- bonding groups is different for AT, TA, GC, and CG base pairs. In the minor groove, there is only one difference in the pattern between AT and GC base pairs.
Distinguishing between features of alternative double-helical structures B-DNA (Watson-Crick DNA) A-DNA Z-DNA
The predominant form of DNA in vivo is B-DNA. But, there is evidence for a role of Z-DNA in vivo: –Z-DNA binding proteins. –Short sections of Z-DNA within a cell are energetically favorable and stable. –Role in regulating gene expression?
A region of Z-DNA is connected to B-DNA through a junction in which one base pair is flipped out, or extruded, from the DNA helix. This process is called base flipping.
DNA can undergo reversible strand separation Significance of complementary base pairing: Fidelity of DNA replication, transcription, and translation. Ability to manipulate in the lab by denaturation, renaturation, and hybridization.
Denaturation or “melting” of DNA Base stacking in duplex DNA quenches the capacity of bases to absorb UV light. Hyperchromicity: As DNA “melts” its absorption of UV light increases. T m (melting temperature): The temperature at which half of the bases in a dsDNA sample have denatured.
Renaturation or “reannealing” of DNA The capacity to renature denatured DNA permits hybridization. Hybridization is the complementary base pairing of strands from two different sources. The rate at which DNA reanneals is a function of the length of the DNA and the initial concentration in the sample.
A DNA renaturation Cot curve C/C 0 = 1/[1 + KC 0 t] The expression C 0 t is called “Cot.” Cot ½ is when renaturation is half completed. A plot of C/C 0 versus C 0 t is called a Cot curve.
Comparison of Cot curves for E. coli and calf thymus DNA The Cot ½ of calf thymus DNA is greater than the Cot ½ of E. coli DNA. Explanation: The larger the genome size, the longer it will take for any one sequence to encounter its complementary sequence.
The E. coli DNA curve resembles an ideal Cot curve. The calf thymus DNA curve is not smooth. Explanation: the rapidly reannealing fraction in the calf DNA represents repeated DNA sequences; the more slowing reannealing fraction represents unique sequences.
2.4 Unusual DNA secondary structures
Slipped structures Occur at tandem repeats Found upstream of regulatory regions
Formation of DNA slipped structures can lead to repeat expansion during DNA replication. A number of hereditary neurological diseases are caused by the expansion of simple triplet repeat sequences.
Cruciform structures Paired stem-loop formations Characterized in vitro for many inverted repeats in plasmids and phage Role in vivo?
Triple helix DNA A third strand of DNA joins the first two to form triplex DNA (intra- or inter-molecular). Favored by purine-pyrimidine stretches with mirror repeat symmetry. The Watson-Crick duplex associates with the third strand through Hoogsteen hydrogen bonds.
Friedreich’s ataxia and triple helix DNA 5′-GAA-3′ trinucleotide repeat expansion in first intron of Friedreich’s ataxia gene (frataxin). Normal individual: 8-30 repeats Friedreich’s Ataxia: 1000 Expanded GAA repeats form triple helix DNA.
2.5 Tertiary structure of DNA
Supercoils form a twisted, 3-D structure which is more favorable energetically. Less stable than relaxed DNA. Negative (left-handed) supercoil: underwound Positive (right-handed) supercoil: overwound Supercoiling of DNA
The strain present within supercoiled DNA sometimes leads to localized denaturation. B-DNA→ Z-DNA transitions may be triggered by negative supercoiling. Topoisomerases are enzymes that introduce transient breaks in DNA strands and release the strain of supercoiling.
What is the significance of supercoiling in vivo? Virtually all DNA within prokaryotes and eukaryotes is negatively supercoiled. Some architectural proteins, induce DNA negative supercoiling upon binding. DNA is restrained when it is supercoiled around DNA-binding proteins, such as in nucleosomes. Unrestrained supercoiled domains are in equilibrium between tension and unwinding of the helix.
DNA supercoiling plays an important role in many processes, such as replication, transcription, and recombination Genome of some viruses: small circle Relaxed circle: reduced activity Negatively supercoiled circle: increased activity Bacterial genome: very large circle Form independent DNA loop domains Eukaryotic genomes: linear Form independent DNA loop domains
Negative supercoiling makes it easier to separate the DNA strands during replication and transcription. The DNA of thermophilic Archaea exists in a positive supercoiled state that protects the DNA from denaturation at high temperatures.