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					  DNARNAProteins

         3.3, 7.1 DNA structure
        3.4, 7.2 DNA replication
3.5, 7.3, 7.4 Transcription and translation
Discovery of Genetic Material: DNA or
protein?
• Biologists knew that genes are located on chromosomes
  (made of DNA and protein)
  – DNA and protein were the candidates for the genetic material
  – Until the 1940s, the case for proteins seemed stronger because
    proteins appeared to be more structurally complex and
    functionally specific.
  – Biologists finally established the role of DNA in heredity
    through studies involving bacteria and the viruses that infect
    them.
Frederick Griffith
• 1928; British medical officer
• Griffith was studying two strains of a bacterium:
  – a pathogenic (disease-causing) strain that cause pneumonia
  – a harmless strain.
• Found that when he mixed a dead version of the pathogenic
  bacteria and harmless bacteria, some living bacterial cells were
  converted to the disease-causing form.
  – Furthermore, all of the descendants of the transformed bacteria
     inherited the newly acquired ability to cause disease.
• Clearly, some chemical component of the dead bacteria could act
  as a “transforming factor” that brought about a heritable change.
   Hershey and Chase
 1952; American biologists
 Experiments showed that DNA is the genetic material of a virus
  (bacteriophage or phage, for short) called T2, which infects E.coli
 T2 consists solely of DNA and protein; DNA-containing head and a
  hollow tail with six jointed fibers extending from it.
 T2 infects bacteria by attaching to the surface with its fibers and
  injecting its hereditary material.
    Raised the question: Is it DNA being passed on or protein?
                        Chase
     Hersheyorandquestion, they devised an experiment to
 To answer protein DNA
  determine what kinds of molecules the phage transferred to E.coli
  during infection
 Used a few relatively simple tools:
   Chemicals containing radioactive isotopes
      To label the DNA and protein in T2
      Used radioactive sulfur and phosphorous
         Sulfur is in proteins. Phosphorous is in DNA.
   A radioactivity detector
   A kitchen blender
   And a centrifuge (device that spins test tubes to separate particles of
    different weights.
   Hershey and Chase
 The Experiment
   1.   First they grew T2 with E.Coli with radioactive sulfur.
   2.   Then, they grew a separate batch of phages in a solution containing
        radioactive phosphorous.
   3.   They allowed the two batches of T2 to infect separate samples of
        nonradioactive bacteria
   4.   Shortly after the onset of infection, they agitated the cultures in a blended to
        shake loose any parts of the phages that remained outside the bacterial cells.
   5.   They then spun the mixtures in a centrifuge. The cells were deposited as a
        pellet at the bottom of the centrifuge tubes, but phages and parts of phages
        being lighter, remained suspended in the liquid.
   6.   The researchers then measured the radioactivity in the pellet and the liquid.
    Hershey and Chase
 The Results
  Found that when the bacteria has been infected with T2 phages
   containing labeled protein, the radioactivity ended up in the liquid
   but not bacteria.
    Result suggested that the phage protein did not enter the cells.
  But when the bacteria had been infected with phages whose DNA
   was tagged, then most of the radioactivity was in the pellet, made up
   of bacteria.
    When these bacteria were returned to liquid growth medium, they soon died
     and lysed and released new phages that contained radioactive phosphorous in
     their DNA but no radioactive sulfur.
    Hershey and Chase
 The Conclusions
  They concluded that T2 injects its DNA into the host cell, leaving
   virtually all its protein outside.
  They demonstrated that it is the injected DNA molecules that cause
   cells to produce additional phage DNA and proteins, making new
   complete phages.
  This indicated that DNA contained the instructions for making
   proteins
  ***THESE RESULTS CONVINCED MOST SCIENTISTS THAT
   DNA IS THE HEREDITARY MATERIAL!!!***
Hershey and Chase
   What is DNA?
 Deoxyribonucleic Acid (DNA)
   Monomers made up of nucleotides:
      Nucleotides consist of:
        A five carbon sugar, deoxyribose
          o Four in it’s ring, one extending above the ring
          o Missing one oxygen when compared to ribose
        Phosphate group
          o Is the source of the “acid” in nucleic acid
        Nitrogenous base (Adenine, Guanine, Cytosine, Thymine)
          o A ring consisting of nitrogen and carbon atoms with various functional groups
             attached
          o Double ring= purines (A and G)
          o Single ring= pyrimidines (T and C)
   Double helix consists of:
     Sugar-phosphate backbone held by covalent bonds
     Nitrogen bases are hydrogen bonded together; A pairs with T and C pairs with G
DNA Structure
The Race to solve the puzzle of
DNA structure
The Race to solve the puzzle of
DNA structure
 A few scientists working on the puzzle trying to determine
  the 3-D structure: Pauling, Wilkins, Franklin, Watson and
  Crick
 Rosalind Franklin observed an X-ray crystallography image
  of the basic shape of DNA
 Watson saw this image, and with just a glance deduced the
  basic shape of DNA to be a helix with a uniform diameter of
  2 nm, with its nitrogenous bases stacked about one-third of a
  nanometer apart.
 The diameter of the helix suggested it was made up of two
  polynucleotide strands=> DOUBLE HELIX!
 The Race to solve the puzzle of
 DNA structure
 Watson and Crick began trying to construct a double helix that
  would conform both Franklin’s data and what was currently
  known about the chemistry of DNA.
 Franklin had concluded that the sugar-phosphate backbones
  must be on the outside of the double-helix, forcing the
  nitrogenous bases to swivel to the interior of the molecule.
 Watson and Crick found that Adenine always paired with
  Thymine, and Guanine and Cytosine, to ensure a uniform
  diameter.
 Complementary base pairing was explained both by the
  physical attributes and chemical bonding of DNA, along with
  data obtained by Chargaff
The Race to solve the puzzle of
DNA structure
 Chargaff’s rules: A always pairs with T and G always pairs
  with C.
 Only apply to base pairing, not the sequence of nucleotides
   The sequence of bases can vary in countless ways, and each gene
    has a unique order of nucleotides, or base sequence
 1962, Watson and Crick received the Nobel Prize for their
  work (Franklin would have received it as well, but she died
  from cancer in 1958; Nobel Prizes are never awarded to the
  deceased)
DNA Replication
“It has not escaped our notice that the specific pairing we have
   postulated immediately suggests a possible copying
   mechanism for the genetic material”
                                        ~Watson and Crick
DNA Replication
 Logic behinds Watson-Crick’s proposal for how DNA is
  copied
   Can be seen by covering one of the strands in the parental DNA
    molecule with a piece of paper: you can determine the bases of
    the covered strand by applying the base-pairing rules: A pair
    with T, and G pairs with C.
   Watson and Crick predicted that a cell applies the same rules
    when copying its genes.
DNA replication- General Overview
    Figure 10.4A Template model for DNA replication
    1. First, the two strands of parental DNA separate, and each
       becomes a template for the assembly of a complementary
       strand from a supply of free nucleotides.
    2. The nucleotides line up one at a time along the template
       strand in accordance with the base-pairing rules
    3. Enzymes link the nucleotides to form the new DNA strands.
    4. Completed new molecules, identical to the parental
       molecule, are known as daughter DNA.
DNA Replication
 Semi-conservative model
   Watson and Crick’s model predicts that when a double helix
    replicates, each of the two daughter molecules will have one old
    strand, which was part of the parental molecule, and one new
    made strand.
   Known as semi-conservative model because half of the parental
    molecule is maintained (conserved) in each daughter molecule.
   Confirmed by experiments performed in the 1950s.
Semi-conservative replication
 DNA Replication- 1. Initiation
 DNA replication begins at specific sites on the double helix
  referred to as origins of replication.
 An enzyme called helicase binds to DNA and separates the
  strands.
   Uses energy from ATP
   Single-strand binding protein (SSB) binds to each strand to prevent
    reannealing
 Replication proceeds in both directions , creating replication
  “bubbles”
 DNA has many origins of replication that can start
  simultaneously, for time efficiency.
 Thousands of bubbles can be present, and eventually all the
  bubbles merge, yielding two completed daughter DNA
  molecules.
DNA Replication- Details
 DNA’s sugar-phosphate backbones run in opposite
  directions.
 Each strands has a 3’ end and a 5’ end.
 The primed number is referring to the carbon atoms of the
  nucleotide sugars.
 At one end of each DNA strand, the sugar’s 3’ carbon atom is
  attached to an –OH group, at the other end, the sugar’s 5’
  carbon has a phosphate group.
DNA Replication- details
DNA Replication - details
 The opposite orientation of the strands is important in DNA
  replication.
 DNA polymerases link DNA nucleotides to a growing
  daughter strand, only to the 3’ end of the strand, never to the
  5’ end.
 Thus, a daughter DNA strand can only grow in the 5’3’
  direction.
DNA Replication- 2. Elongation
 Replication fork forms:
   Partial opening of a DNA helix to form two single strands that
    has a fork appearance.
   Primers required by DNA polymerases during replication are
    synthesized by an enzyme RNA primase.
     Enzyme is an RNA polymerase that synthesizes short stretches of RNA
      that function as primers for DNA polymerases. Later on, the RNA primer
      is removed and replaced with DNA.
   DNA polymerase removes primers and adds DNA
    nucleotides.
     We will be looking at two types of DNA Polymerase: DNA polymerase
      III and DNA polymerase I
DNA Replication- Elongation
  one of the daughter strands can be synthesized in one
   continuous fashion from an initial primer, working toward the
   forking point of the parental DNA. leading strand
    Only a single priming event is required, and then the strand can be
     extended indefinitely by DNA polymerase III.
DNA Replication- Elongation
  The other daughter strand polymerase molecules must work
  outward from the forking point, is synthesized in short pieces as
  the fork opens up in a discontinous fashion involving multiple
  priming events. lagging strand
    DNA Polymerase III adds nucleotides in 5’3’ direction.
     Primers get removed via and replaced by DNA via DNA polymerase I
    Fragments formed are called Okazaki fragments
    DNA ligase links (ligates) the pieces together into a single DNA strand.
DNA Replication- Termination
 At the completion DNA replication, you end up with two
  identical strands of DNA.
 Each DNA molecule contains one original parent strand, and
  one new daughter strand. Semi conservative
  replication
DNA replication animation
 http://www.wiley.com/college/pratt/0471393878/studen
 t/animations/dna_replication/index.html
DNA Replication
 Key enzymes:
   Helicase: unwinds the double helix
   Primase: synthesizes RNA primers
   SSB: stabilizes single-stranded regions; prevents reannealing
   DNA polymerase III- synthesized DNA
   DNA polymerase I- erases primer and fills gaps
   DNA ligase- joins the ends of DNA segments; DNA repair
DNA replication
 Process is not only fast but also amazingly accurate
 Typically, only about one DNA nucleotide per billion is
  incorrectly paired
 DNA polymerase carry out a proofreading step that quickly
  removes nucleotides that have base-paired incorrectly
 DNA polymerases and DNA ligase are also involved in
  repairing damaged DNA by harmful radiation or toxic
  chemicals
 Ensure that all somatic cells in a multicellular organism carry
  the same genetic information.
Protein synthesis: overview
 DNA inherited by an organism specifies traits by dictating
  the synthesis of proteins.
 However, a gene does not build a protein directly; it
  dispatches instruction in the form of RNA, which in turn
  programs protein synthesis.
 Message from DNA in the nucleus of the cell is sent on RNA
  to protein synthesis in the cytoplasm.
 Two main stages:
   Transcription
   Translation
Protein Synthesis: Overview
 Two main stages:
   Transcription
     The transfer of genetic information from DNA into an RNA molecule
     Occurs in the eukaryotic cell nucleus
     RNA is transcribed from a template DNA strand
   Translation
     Transfer of the information in RNA into a protein.
   Transcription
 Details:
   1. Initiation-
     Promoter is the nucleotide sequence on DNA that marks where
       transcription of a gene begins and ends; “start” signal
     Promoter serves as a specific binding site for RNA polymerase and
       determines which of the two strands of the DNA double helix is used as the
       template.
     Specific nucleotide sequence at promoter is TATAAA
     Called the “TATA box”; located 25-35 base pairs before the transcription
       start site of a gene
     TATA box is able to define the direction of transcription and also indicates
       the DNA strand to be read
     Proteins called transcription factors can bind to the TATA box and recruit
       RNA polymerase; it has a regulatory function
     Note:TATA box is found upstream of start site and thus is NOT transcribed by RNA
       polymerase
Transcription
  Elongation-
    RNA elongates
    As RNA synthesis continues, the RNA strand peels away from its DNA
     template, allowing the two separated DNA strands to come back together
     in the region already transcribed.
Transcription
 3. Termination-
   RNA polymerase reaches a sequence of bases in the DNA template called
     a terminator.
   Signals the end of the gene; at that point, the polymerase molecule
     detaches from the RNA molecule and the gene.
   mRNA (messenger RNA) or “transcript” exits the nucleus via the nuclear
     pores and enter the cytoplasm
Transcription animation
 http://www-
 class.unl.edu/biochem/gp2/m_biology/animation/gene/ge
 ne_a2.html
     RNA processing
 Before mRNA leaves the nucleus, it is modified or processed.
 1. addition of extra nucleotides to the ends of the transcript
   Include addition of a small cap (a single G nucleotide) at one end and
    a long tail (a chain of 50 to 250 A’s) at the other end
   Cap and tail facilitate the export of the mRNA from the nucleus,
    protecting the transcript from attack by cellular enzymes, and help
    ribosomes bind to the mRNA
   Cap and tail are NOT translated into protein.

     http://vcell.ndsu.edu/animations/mrnaprocessing/movie.htm
 RNA processing
 2. RNA splicing
   Cutting-and-pasting process catalyzed by a complex of proteins
    and small RNA molecules, but sometime the RNA transcript
    itself catalyzes the process.
   Introns
      “intervening sequences”; internal noncoding regions
      Get removed from transcript before it leaves nucleus
   Exons
     Coding regions; parts of a gene that are expressed as amino acids
     Joined to produce an mRNA molecule with a continuous coding sequence
     Cap and tail are considered parts of the first and last exons, although are not
      translated into proteins.
 http://student.ccbcmd.edu/biotutorials/protsyn/exon.html
RNA processing
More animations
  http://www.pbs.org/wgbh/aso/tryit/dna/protein.html
  http://www.wisc-
  online.com/objects/index_tj.asp?objID=AP1302
 Translation
 A typical gene consists or hundreds or thousands of
  nucleotides in a specific sequence, which get transcribed onto
  mRNA.
 Translation is the conversion of nucleic acid language into
  polypeptide language
 There are 20 different amino acids.
   A cell has a supply of amino acids in cytoplasm, either obtained
    by food or made from other chemicals.
 Flow of information from gene to protein is based on a
  triplet code: genetic instructions for the a.a. sequence of a
  polypeptide chain are written in DNA and mRNA as a series
  of three-base pairs, or codons.
Translation- tRNA
 To convert the codons of nucleic acids on mRNA to the
  amino acids of proteins, a cell employs a molecular
  interpreter, called transfer RNA (tRNA)
 tRNA molecules are responsible for matching amino acids to
  the appropriate codons to form the new polypeptide.
 tRNA’s unique structure enables it to be able to:
   1. pick up the appropriate amino acids
   2. recognize the appropriate codons in the mRNA
Translation- tRNA
 tRNA is made of a single strand of RNA consisting of about
  80 nucleotides
 By twisting and folding upon itself, it forms several double-
  stranded regions in which short stretches of RNA base-pair
  with other stretches.
 at one end of the folded molecule contains a special triplet of
  bases called an anticodon.
   Complementary to a codon triplet on mRNA
   Anticodon recognizes a particular codon triplet on mRNA
 At the other end of the tRNA molecule is a site where an
  amino acid can attach.
Translation- tRNA
Translation- tRNA
 Each amino acid is joined to the correct tRNA by a specific
  enzyme.
 Each enzyme specifically binds one type of amino acid to all
  tRNA molecules that code for that amino acid, using a
  molecule of ATP as energy to drive the reaction.
 The resulting amino acid-tRNA complex can furnish its
  amino acid to a growing polypeptide chain.
     Translation- rRNA
 Ribosomal RNA (rRNA)
   Organelle in the cytoplasm that coordinates the functioning of
    mRNA and tRNA and actually makes polypeptides.
   Consists of two subunits: large and small
   Each ribosome has a binding site for mRNA, and three binding sites
    for tRNA.
     E site
        Removes tRNA from ribosome
     P site
       Holds the growing polypeptide
     A site
       Obtains new amino-acid-tRNA
   Ribosome holds tRNA and mRNA molecules close together, allowing
    the amino acids carried by the tRNA molecules to be connected into
    a polypeptide chain.
 Translation- Steps
 Can be divided into same three phases: initiation, elongation,
  and termination.
 1. Initiation
   Brings together the mRNA, a tRNA bearing the first amino acid,
    and the two subunits of a ribosome.
   Role is to establish exactly where translation will begin, ensuring
    the mRNA codons are translated into the correct sequence of
    amino acids.
Translation
  1. Initiation (continued…)
  Two steps:
    1. an mRNA binds to a small ribosomal subunit. A special initiator tRNA
     binds to the specific codon, called the start codon, where translation
     begins on mRNA.
       Initiator tRNA carries the amino acid Methionine (Met); its anticodon
        UAC binds to the start codon, AUG
    2.A large ribosomal subunit binds to the smaller one, creating a function
     ribosome. The initiator tRNA fits into tRNA binding site (P site) on the
     ribosome. A site is vacant and ready for the next amino-acid carrying
     tRNA.
 2. Elongation
   Once initiation is complete, amino acids are added one by one to
    the first amino acid. Each addition occurs in a three step process:
     1. codon recognition
         The anticodon of an incoming tRNA carrying an amino acid, pairs with
          the mRNA codon in the A site of the ribosome
     2. peptide bond formation
       Polypeptide separates from the tRNA to which it was bound (P site) and
          attaches by a peptide bond to the amino acid carried by the tRNA in the A
          site.
       The ribosome catalyzes formation of the bond.
     3. translocation
       P site tRNA, moves to the E site and leaves the ribosome.
       The ribosome then translocates (moves) the tRNA in the A site, with its
          attached polypeptide, to the P site.
       Codon and anticodon remain bonded, and the mRNA and tRNA move as
          a unit
       Movement brings into the A site the next mRNA codon to be translated,
          and the process begins again at step 1.
 Termination
   Elongation continues until a stop codon reaches the ribosome’s
    A site.
   Stop codons- UAA, UAG, and UGA, do not code for amino
    acids but instead act as signal to stop translation.
   The completed polypeptide is released from the last tRNA and
    exits the ribosome, which then splits into its separate subunits.
Translation Animation
 http://www-
 class.unl.edu/biochem/gp2/m_biology/animation/gene/ge
 ne_a3.html
 Polysome
Several ribosomes can translate an mRNA at the same time,
forming what is called a polysome.
Peptide Bond Formation
Free ribosomes vs. bound
ribosomes
 Free ribosomes
   Found in cytoplasm
   Synthesize proteins for use primarily within the cell
 Bound ribosomes
   Found on rough ER
   Synthesize proteins primarily for secretion or for lysosomes
Free ribosomes vs. bound
ribosomes
After protein synthesis…
 Each polypeptide coils and folds, assuming a 3-D shape, its
  tertiary structure.
 Several polypeptides may come together, forming a protein
  with quaternary structure.
 Overall significance:
   Process whereby genes control the structures and activities of
    cells
   The way genotypes determine phenotypes; proteins made from
    the original DNA nucleotides determine the appearance and
    capabilities of the cell and organism!
Mutations
 Mutation is any change in the nucleotide sequence of DNA.
 Can involve large regions of a chromosome or just a single
  nucleotide pair, as in sickle cell disease
   In one of the two kinds of polypeptides in the hemoglobin
    protein, the sickle-cell individual has a single different amino
    acid.
   This small difference is caused by a change of a single
    nucleotide in the coding strand of DNA. Only ONE base pair!
Mutations
 Two general categories:
   Base substitution
     Also known as a point mutation
     Replacement of one nucleotide with another.
     Depending on how the base substitution is translated, it can result in no
      change in the protein (due to redundancy of genetic code), an insignficant
      change, or a change that significantly affects the individual.
        Occasionally, it leads to an improved protein that enhances the success
         of the mutant organism and its descendants.
        More frequently, its harmful.
         o May cause changes in protein that prevent it from functionally
            normally.
         o If stop codon is a result of mutation and protein is shortened, it may
            not function at all.
Mutations
 Base insertions or deletions
   Also known as frameshift mutation
   Often has a disastrous effect
   Adding or subtracting nucleotides may result in an alteration of
    the reading frame of the message
      all the nucleotides that are “downstream” of the insertion or deletion will
       be regrouped into different codons.
      Result will most likely by a nonfunctional polypeptide
Mutations
 What causes mutations?
   Mutagenesis, or the production of mutations, can occur in a
    number of ways.
   Errors that occur during DNA replication or recombination are
    called spontaneous mutations.
   Mutagen, a physical or chemical agent that causes mutations
     Physical mutagen: high-energy radiation, such as X-rays and UV light
     Chemical mutagen: consists of chemicals that are similar to normal DNA
      bases pair incorrectly.
Mutations
 Can also be helpful both in nature and in the laboratory.
 It is because of mutations that there is such a rich diversity of
  genes in the living world, that make evolution by natural
  selection possible.
 Also essential tools for geneticists.
   Whether naturally occurring or created in the laboratory,
    mutations create the different alleles needed for genetic
    research.

				
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