Biochemistry

Lecture 17: Nucleic Acid Structures

Assigned reading in Campbell: Chapter 7

Take the Review Quiz on Lecture 17 concepts.

Class handouts (PDF) for this lecture: 1. Backbone & Bases and 2. Base Pairs
Nucleic Acid Structures is a page of JPEG images to accompany this lecture.

1. DNA and RNA are Polynucleotides

1. The phosphodiester backbone is comprised of deoxyribose (DNA) or ribose (RNA) sugars bridged by phosphates between the 3' and 5' positions of the sugars.
   1. The phosphates are always ionized (pK_a~1).
   2. Nucleic acids are polyanions.
   3. Note:
      a) the polarity, i.e. 5' --> 3';
      b) the 6 - 7 bonds with free rotation in polynucleotides.
2. The sequence of nucleotides encodes the information in DNA.
   1. Chargaff's rules: A = T & G = C for double-stranded DNA.
   2. U replaces T in RNA.
   3. Nucleotide bases can be modified.
      a) A and C are sometimes methylated in DNA.
      b) Dozens of modified bases are found in tRNA.

2. Double Helical Structures

1. Watson-Crick structure: B-DNA
   1. The helix is right-handed; the chains are antiparallel.
   2. 3.4 Å/base pair; 10 bp/turn.
   3. Major & minor grooves are the "front" and "back" surfaces; the helix interior is filled with stacked bases.
   4. Bases are connected to the C1' of the sugar: glycosidic bond.
2. A-DNA and double-helical RNA.
   1. Right-handed helix; antiparallel chains.
   2. 2.6 Å/base pair; 11 bp/turn.
   3. Deep and shallow grooves are the "front" and "back" surfaces.
   4. RNA cannot adopt the B-form (the 2'-OH interferes).
3. Additional comments on nucleic acid helices.
   1. Base pairs are in identical environments along the helix.
   2. Helical features differ from their ideal (average) values.
      a) Sequence affects local structure.
      b) Twist and tilt vary over time (on a nsec time scale).
      c) Ligands alter DNA & RNA structures.
   3. Z-DNA is not (yet) a significant structure, biologically.
   4. RNA displays more diverse structures.
      a) hairpins: double helical stem and loop.
      b) internal loops: within double helical segments.
      c) tRNA is a globular molecule.
4. Genomic DNAs are large molecules.
   1. Eschericia coli: 4.7*10⁶ bp; ~ 1 mm contour length.
   2. Human: 2.9*10⁹ bp; ~ 1 m contour length.

3. Forces Stabilizing Nucleic Acid Structures.

1. DNA can be reversibly denatured ("melting").
   1. Cooperative transition from helix --> random coils; monitor DA₂₆₀.
   2. T_m (the midpoint) increases with G + C content.
   3. T_m increases with increased salt concentration.
2. Base pairing
   1. Watson-Crick H-bonding is only a minor contribution to stability.
   2. Base pairing is essential for specificity.
3. Base stacking is the major contribution to helix stability.
   1. Planar aromatic bases overlap geometrically and electronically.
   2. Stacking is more than a hydrophobic effect.
      a) H₂O is excluded between base pairs, but fills the grooves.
      b) But, the magnitude and nature of stacking differs from the hydrophobic effect in proteins.

4. Nucleic Acid Fractionation

1. DNA & RNA are precipitated with >67% ethanol.
2. Affinity chromatography is used to purify specific nucleic acids.
   1. Poly (T) columns bind the poly (A) portion of eukaryotic mRNAs.
   2. In general: an immobilized oligomer will bind its complement in a complex mixture containing other sequences.
3. Electrophoresis
   1. PAGE (polyacrylamide gel electrophoresis) for small nucleic acids.
   2. Agarose gels are used for large (and very large) nucleic acids.
   3. Detection
      a) visualization with fluorescent intercalators.
      (Several aromatic drugs and dyes, when bound to DNA, can slip between the stacked base pairs of DNA, a process called intercalation.)
      b) autoradiography of ³²P-labelled nucleic acids.
   4. log (length in bp) vs. migration is linear.
      (cf. SDS PAGE of proteins)
4. Ultracentrifugation
         - Rarely used since the advent of PCR. -

5. Supercoiled DNA

1. DNA is underwound in the cell, causing supercoiling of the helix.
   1. Eukaryotes: DNA is wrapped around histone octamers.
   2. Prokaryotes: DNA is under torsional tension.
2. Supercoiling is maintained by DNA gyrase + ATP hydrolysis.
3. Supercoiling is necessary for the (essential) compaction of genomes.
4. Supercoiling is a reservoir of energy for enzymes that must unwind the DNA double helix for their functions.

Please note: The following topics are not covered in Campbell, but keep in mind the descriptions listed here and covered in lecture.

6. Nucleic Acid Sequencing

1. Everyone knows how this is done, in principle, right?
   1. Chemical method.
   2. Chain terminator method. Related to Lecture 18, DNA Replication.
2. Here's an update on genome sequencing projects.
   1. Bacteria: (as of 1998) >20 genomes; ~5 Mbp each; ~3000 genes each.
   2. Yeast: (1996) entire genome; 12 Mbp; ~6200 genes.
   3. Worm: (1998) entire genome; 97 Mbp; ~19,100 genes.
   4. Fruit fly: (2000) entire genome; 120 Mbp; ~13,600 genes.
   5. Human: (2000) 3,000 Mbp; ~140,000 genes.
          [Note: This is gratuitous information; do not memorize it!]

7. Chemical Synthesis of Oligonuceotides

1. Solid-phase synthesis of DNA and RNA is completely automated at hundreds of centers and commercial firms.
2. Almost no one knows (or remembers) the chemical details of how it is done.
3. The important point is to write the desired sequence correctly on the order form that is FAXed to the company, because whatever you write down shows up in the mail about 2-3 days later.

8. Molecular Cloning

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