DNA replication and transcription

• DNA Replication


  The process by which a DNA molecule is copied to produce two identical DNA molecules
• Transcription

  The process by which the information in a strand of DNA is copied to make a complementary strand of RNA
• The information content of genes is in the specific sequences of nucleotides
• The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins
• DNA must be replicated for cells to divide
• Gene expression, the process by which DNA directs protein synthesis, includes two stages

  • Transcription
  • Translation
• Genetic material


  DNA, RNA, protein
• The discovery of DNA

  1. 19th century: observations of Mendel and others indicate the genetic material is contained in cells

  2. 1928: Frederick Griffith demonstrated the transmission of genetic instructions by a process we now call the "transformation principle"

  3. 1944: Avery, MacLeod and McCarty suggest DNA is the "transforming factor", and not proteins or other materials

  4. 1952: Hershey and Chase proved that DNA was the genetic material in bacteriophage

  5. 1848, Wilhelm Hofmeister, a German botanist, observed that cell nuclei resolve themselves into small, rodlike bodies during mitosis. These bodies were found to absorb certain dyes and so came to be called chromosomes (coloured bodies)
• Smooth (S) and rough (R) S. pneumoniae

  • S phenotype due to a polysaccharide capsule
  • R bacteria were harmless, but the S bacteria caused disease
  • Heat-killed S cells were also harmless
  • When heat killed S bacteria were mixed with R bacteria, the injected mice got sick and died
• Nonvirulent strain of bacteria can be transformed into a virulent strain of bacteria by an unknown molecule from the virulent cells
• Watson & Crick: Built models of a double helix to conform to the X-rays produced by Franklin
• Watson & Crick's model of DNA

  • Outer sugar-phosphate backbone, with the nitrogenous bases paired in the molecule's interior
  • Backbones were antiparallel (their subunits run in opposite directions)
• Base pairing

  • Adenine (A) paired only with thymine (T), guanine (G) paired only with cytosine (C)
  • Explains the observation that, in any organism, the amount of A = T, and the amount of G = C
  • Purine + pyrimidine: width consistent with X-ray data
• Nucleotides

  • Serve as the monomer units for forming the nucleic acid polymers of DNA and RNA
  • Composed of three subunit molecules: nitrogenous base, five-carbon sugar, and at least one phosphate group
  • A five-carbon sugar molecule attached to a nitrogenous base is called a nucleoside
• The 2 types of nucleic acid

  • DNA - deoxyribonucleic acid
  • RNA - ribonucleic acid
• DNA

  • Replicates and stores genetic information
  • Consists of two strands, arranged in a double helix, made up of nucleotide subunits
  • Longer polymers than RNA. E.g. a chromosome is a single DNA molecule, millions of bases
  • The sugar in DNA is deoxyribose
  • The bases in DNA are Adenine ('A'), Thymine ('T'), Guanine ('G') and Cytosine ('C')
• RNA

  • Converts the genetic information contained within DNA to a format used to build proteins
  • Only has one strand, but like DNA, is made up of nucleotides
  • Variable in length, but typically quite short. E.g. mRNAs 100's – 1000's bases
  • The sugar in RNA is ribose
  • The bases in RNA are Adenine ('A'), Uracil ('U'), Guanine ('G') and Cytosine ('C')
• Structural overview - DNA vs. RNA

  • RNA ss, DNA ds
  • RNA ribose, DNA deoxyribose
  • RNA U, DNA T
• Uracil is used in RNA because

  • Less energetically expensive than T
  • T susceptible to oxidation outside of nucleus
  • U less stable but short-lived
• Structure of a DNA strand

  • Polynucleotide chains have nitrogenous bases linked to a sugar-phosphate backbone
  • Nucleotides are linked by phosphodiester bonds (C-O-P-O) to form a DNA strand
  • Phosphodiester bonds of the DNA give the polarity of the DNA strand (5' phosphate and 3' hydroxyl end)
• Structure of the DNA double helix

  • Hydrogen bonds between the bases of the opposite strands and base stacking of bases within a strand contribute to the stability of DNA double helix
  • Phosphodiester bond of the backbone is also relatively stable
  • dsDNA can withstand stress like heat and pH
• DNA replication


  • The basic principle of replication is base pairing to a template strand
  • Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication
  • In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules
• DNA replication - semi conservative


  Semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand ("conserved" from the parent molecule) and one new strand
• Meselson & Stahl - 1958

  1. Labelled nucleotides of parent strands with a heavy isotope of nitrogen

  2. First replication produced a band of hybrid DNA, eliminating the conservative model

  3. Second replication produced both light and hybrid DNA, eliminating the dispersive model

  4. Supported the semiconservative model: each parent strand acting as a template for synthesis of a complementary strand
• Process of DNA replication

  Replication of DNA is undertaken by a complex of enzymes that separate the parental strands and synthesize the daughter strands (more than a dozen enzymes and other accessory proteins participate in DNA replication)
• Replication begins at origins of replication (ori)

  1. The two DNA strands are separated, opening up a "replication bubble"

  2. Replication proceeds in both directions from the ori
• Theta replication is commonly used for replication of circular chromosomes (bacteria)
• Linear DNA replication takes place in eukaryotic chromosomes
• New DNA strand elongation

  • Each nucleotide that is added to a growing DNA strand is part of a dNTP molecule
  • As each monomer of dNTP joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate
• Overview of DNA replication

  1. Primase produces an RNA chain called the primer

  2. A primer is short (5–10 nucleotides long)
• DNA replication


  1. Replication of DNA is undertaken by a complex of enzymes that separate the parental strands and synthesize the daughter strands

  2. (more than a dozen enzymes and other accessory proteins participate in DNA replication)
• Replication begins at origins of replication (ori)


  1. Replication begins at specific sites called origins of replication

  2. The two DNA strands are separated, opening up a "replication bubble"

  3. Replication proceeds in both directions from the ori
• Theta replication


  Commonly used for replication of circular chromosomes (bacteria)
• Linear DNA replication


  Takes place in eukaryotic chromosomes
• New DNA strand elongation

  1. Each nucleotide that is added to a growing DNA strand is part of a dNTP molecule

  2. As each monomer of dNTP joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate
• How does DNA replication start?

  1. Primase produces an RNA chain called the primer

  2. A primer is short (5–10 nucleotides long): the 3′ end serves as the starting point for the new DNA strand

  3. It attaches to the template and adds RNA nucleotides one at a time with the parental DNA as a template

  4. DNA polymerase catalyses the elongation of new DNA from the primer

  5. The rate of elongation can be up to 1000 nucleotides per second in bacteria and mammalian cells
• E. coli


  4.5 mbp <40min
• Human


  3 bbp ~60min
• DNA strands always elongated in 5' to 3' direction
• DNA polymerases require a 3′–OH priming end to initiate DNA synthesis