Chapter 19 (part 2)

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Chapter 19 (part 2)

• Nucleic Acids

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DNA

• 1o Structure - Linear array of nucleotides
• 2o Structure double helix
• 3o Structure - Super-coiling, stem-loop formation
• 4o Structure Packaging into chromatin

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Determination of the DNA 1o Structure (DNA
Sequencing)

• Can determine the sequence of DNA base pairs in
  any DNA molecule
• Chain-termination method developed by Sanger
• Involves in vitro replication of target DNA
• Technology led to the sequencing of the human
  genome

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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
• 4 separate rxns
• Each reaction mixture contains dATP, dGTP, dCTP
  and dTTP
• Each reaction also contains a small amount of one
  dideoxynucleotide (ddATP, ddGTP, ddCTP and
  ddTTP).
• Each of the 4 dideoxynucleotides are labeled with
  a different fluorescent dye.
• Dideoxynucleotides missing 3-OH group. Once
  incorporated into the DNA chain, chain elongation
  stops)

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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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DNA Secondary structure

• DNA is double stranded with antiparallel strands
• Right hand double helix
• Three different helical forms (A, B and Z DNA.

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Comparison of A, B, Z DNA

• A right-handed, short and broad, 2.3 A, 11 bp
  per turn
• B right-handed, longer, thinner, 3.32 A, 10 bp
  per turn
• Z left-handed, longest, thinnest, 3.8 A, 12 bp
  per turn

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A-DNA
B-DNA
Z-DNA
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Z-DNA

• Found in GC-rich regions of DNA
• G goes to syn conformation
• C stays anti but whole C nucleoside (base and
  sugar) flips 180 degrees

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DNA sequence Determines Melting Point

• Double Strand DNA can be denatured by heat (get
  strand separation)
• Can determine degree of denturation by measuring
  absorbance at 260 nm.
• Conjugated double bonds in bases absorb light at
  260 nm.
• Base stacking causes less absorbance.
• Increased single strandedness causes increase in
  absorbance

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DNA sequence Determines Melting Point

• Melting temperature related to GC and AT
  content.
• 3 H-bonds of GC pair require higher temperatures
  to denture than 2 H-bonds of AT pair.

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DNA 3o Structure

• Super coiling
• Cruciform structures

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Supercoils

• In duplex DNA, ten bp per turn of helix (relaxed
  form)
• DNA helix can be over-wound.
• Over winding of DNA helix can be compensated by
  supercoiling.
• Supercoiling prevalent in circular DNA molecules
  and within local regions of long linear DNA
  strands
• Enzymes called topoisomerases or gyrases can
  introduce or remove supercoils
• In vivo most DNA is negatively supercoiled.
• Therefore, it is easy to unwind short regions of
  the molecule to allow access for enzymes

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Each super coil compensates for one or turn
of the double helix
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• Cruciforms occur in palindromic regions of DNA
• Can form intrachain base pairing
• Negative supercoiling may promote cruciforms

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DNA and Nanotechnology
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DNA and Nanotechnology
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DNA 4o Structure

• In chromosomes, DNA is tightly associated with
  proteins

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Chromosome Structure

• Human DNAs total length is 2 meters!
• This must be packaged into a nucleus that is
  about 5 micrometers 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
• major histone proteins H1, H2A, H2B, H3 and H4
• Histone octamers are major part of the protein
  spools
• Nonhistone proteins are regulators of gene
  expression

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• 4 major histone (H2A, H2B, H3, H4) proteins for
  octomer
• 200 base pair long DNA strand winds around the
  octomer
• 146 base pair DNA spacer separates individual
  nucleosomes
• H1 protein involved in higher-order chromatin
  structure.
• W/O H1, Chromatin looks like beads on string

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Solenoid Structure of Chromatin
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RNA

• Single stranded molecule
• Chemically less stable than DNA
• presence of 2-OH makes RNA more susceptible to
  hydrolytic attack (especially form bases)
• Prone to degradation by Ribonucleases (Rnases)
• Has secondary structure. Can form intrachain base
  pairing (i.e.cruciform structures).
• Multiple functions

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Type of RNA

• Ribosomal RNA (rRNA) integral part of ribosomes
  (very abundant)
• Transfer RNA (tRNA) carries activated amino
  acids to ribosomes.
• Messenger RNA (mRNA) endcodes sequences of
  amino acids in proteins.
• Catalytic RNA (Ribozymes) catalzye cleavage of
  specific RNA species.

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RNA can have extensive 2o structure