Topic 7 - Nucleic Acids

• Nucleosomes


  • Unlike most prokaryotic DNA which is referred to as 'naked', eukaryotic nuclear DNA is associated with proteins called histones (to form chromatin)
  • Histones package the DNA into structures called nucleosomes
  • The nucleosome consists of a strand of DNA coiled around a core of eight histone proteins (octamer) to form a bead-like structure
  • DNA takes two turns around the histone core and is held in place by an additional histone protein
  • The DNA molecule continues to be wound around a series of nucleosomes to form what looks like a 'string of beads'
  • Nucleosomes help to supercoil the DNA, resulting in a compact structure which saves space within the nucleus
  • Nucleosomes also help to protect DNA and facilitate movement of chromosomes during cell division

  
• Supercoiling


  An analogy for supercoiling is twisting an elastic band repeatedly until it forms additional coils
• Nucleosomes


  Can be tagged with proteins to promote or suppress transcription
• DNA is wrapped around a series of nucleosomes. Nucleosomes coil tightly around each other to form the chromosome structure.

  
• Rosalind Franklin's X-ray diffraction


  • Provided crucial evidence that DNA is a double helix
  • Franklin's work was instrumental to Crick and Watson's model as the diffraction patterns indicated that DNA had a double-helical structure

  
• 1. ray diffraction


  1. Directing a beam of X-rays onto the molecule being studied

  2. X-rays have a shorter wavelength and higher energy than visible light

  3. The short wavelength allows X-rays to pass through the molecule, interacting with any electrons within the atoms

  4. The interaction causes X-rays to scatter (diffraction) at angles that indicate the arrangement of atoms

  5. The scattering pattern can be recorded on a film (similar to having an X-ray of a bone), with dark marks appearing where the X-rays strike the film

  6. Rotating the sample allows for the three-dimensional molecular structure to be studied

  
• Franklin was able to refine her methods and produce a clear diffraction pattern of DNA
• Using mathematical techniques and available knowledge about DNA, Franklin deduced that DNA strands were helices, the pitch of the helix, the distance between nucleotides, phosphates are located on the outside of the molecule, and DNA was double stranded

  
• Semi-conservative DNA replication


  Half of the original DNA molecule is kept (conserved) in each of the two new DNA molecules

  
• Crick and Watson's theory of semi-conservative DNA replication was later proven by Meselson and Stahl

  
• Leading strand


  • Made continuously, following the fork as it opens
• Lagging strand


  • Made discontinuously, in short fragments, away from the fork
• DNA replication


  1. Helicase unwinds the DNA double helix at the replication fork by flattening out its helical structure

  2. Helicase then causes the hydrogen bonds between the two strands to break

  3. DNA gyrase releases the strain within the supercoiled areas to allow helicase access to the helix

  4. Single-stranded binding proteins keep the separated strands apart whilst the template strand is copied

  5. DNA primase generates a short RNA primer on the template strands

  6. DNA polymerase III starts replication next to the RNA primer linking nucleotides in a 5' to 3' direction to form a new strand

  7. DNA polymerase I removes the RNA primers on the leading and lagging strands and replaces it with DNA

  8. DNA ligase joins up the Okazaki fragments by catalysing the formation of sugar-phosphate bonds

  
• DNA polymerase only works in a 5' to 3' direction, adding nucleotides to the 3' end

  
• Non-coding regions of DNA

  • Involved in the control of gene expression by enhancing or suppressing transcription
  • Can produce functional RNA molecules like transfer RNA (tRNA)
  • Introns are non-coding sequences of DNA found within genes of eukaryotic organisms
  • Telomeres are regions of repeated nucleotide sequences at the end of chromosomes that provide protection during cell division

  
• In humans only 1.5% of the genome contains coding sequences
• DNA profiling


  • Enables individuals to be identified based on their DNA profiles
  • Short, non-coding regions of DNA called variable number tandem repeats (VNTRs) are analysed
  • The frequency that VNTRs are repeated is unique between different individuals
  • VNTRs are inherited and are similar in close relatives but different in unrelated people
  • Monozygotic (identical) twins inherit identical VNTRs so can't be differentiated through profiling
  • To compare the respective DNA profiles of individuals, different regions of DNA containing the VNTRs can be excised with restriction enzymes or amplified by PCR (Polymerase Chain Reaction)
• Introns


  Non-coding regions of DNA that must be removed before translation can occur

  
• DNA profiling


  Identifying individuals based on their unique DNA profiles
• DNA profiling


  • Can be used in forensic investigations or paternity testing
  • Analyses short, non-coding regions of DNA called variable number tandem repeats (VNTRs)
  • The frequency that VNTRs are repeated is unique between different individuals
  • VNTRs are inherited and are similar in close relatives but different in unrelated people
  • Monozygotic (identical) twins inherit identical VNTRs so can't be differentiated through profiling
• Comparing DNA profiles


  1. Different regions of DNA containing the VNTRs can be excised with restriction enzymes or amplified by PCR

  2. The VNTR region for individuals will be a different size as they have different numbers of repeats

  3. The resulting restriction fragment or amplified DNA will also be a different size

  4. Different sized fragments will generate a unique DNA profile in gel electrophoresis

  
• Dideoxynucleotides


  Modified nucleotides that lack the 3'-hydroxyl group so cannot form a covalent bond with the next nucleotide, preventing elongation of the nucleotide chain

  
• Chain-termination method


  Uses dideoxynucleotides to stop DNA replication in preparation of samples for base sequencing

  
• The chain-termination method was developed by Frederick Sanger in 1977
• DNA sequencing using chain-termination method

  1. DNA sample of interest is used as a template in chain-termination PCR

  2. Deoxynucleotides and fluorescently-labelled dideoxynucleotides are used

  3. In the extension step of PCR, DNA polymerase will incorporate deoxynucleotides

  4. If a dideoxynucleotide is randomly incorporated, extension stops

  5. The fragments can separated by size in gel electrophoresis

  6. The fluorescent marker corresponds to a particular 'terminator' nucleotide and can be visualised

  7. This allows the base sequence to be built up one base at a time

  
• High-throughput sequencing methods have enabled rapid sequencing of organism genomes
• Hershey and Chase experiment

  Showed that DNA, not protein, is the heritable material responsible for carrying genetic information
• Hershey and Chase experiment

  1. Bacteria grown in media with radioactive sulfur or phosphorus were infected with viruses

  2. Progeny viruses contained either sulfur-labelled proteins or phosphorus-labelled DNA

  3. Bacteria were separated from viruses and only the bacteria infected by phosphorus-labelled viruses were radioactive

  4. This suggested DNA, not protein, was transferred to bacteria and is the hereditary material

  
• Molecular visualisation software


  Allows researchers to analyse macromolecules and study interactions between them
• Macromolecules like protein, DNA, RNA and complex carbohydrates can be visualised as 3-D structures using molecular visualisation software
• The Protein Data Bank (PDB) is a repository for molecular visualisation software
• Nucleosome


  • DNA double helix makes two loops around a histone octamer core
  • The tails of each histone protein can be chemically modified to help regulate gene expression

  
• Promoter


  A non-coding DNA sequence that acts as the binding site for RNA Polymerase during the initiation of transcription

  
• Regulatory transcription factors


  • Activators bind to enhancer sequences and increase the rate of transcription
  • Repressors bind to silencer sequences and decrease or block transcription

  
• General transcription factors


  Bind directly to the promoter to help initiate transcription and allow RNA polymerase to attach

  
• Regulation of gene expression in prokaryotes

  1. Repressor proteins bind to DNA near the promoter to block RNA polymerase from accessing that section of the genome

  2. When the inducer (e.g. lactose) is present, it binds to the repressor allowing RNA polymerase to begin transcription

  
• Regulation of gene expression ensures that only the genes required are being expressed in the correct cells, at the correct time and to the right level

  
• Gene expression and lactose-degrading enzyme production


  1. Genes are expressed

  2. Lactose-degrading enzymes are produced
• Lactose metabolism


  1. Lactose can be broken down

  2. Used for energy generation
• Regulation of gene expression

  1. When lactose is metabolised, genes are repressed again

  2. Mechanism of negative feedback ensures cell's resources are not wasted making unnecessary proteins