From DNA to Protein: Genotype to Phenotype

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					12
From DNA to Protein:
Genotype to Phenotype
12 From DNA to Protein: Genotype to Phenotype

    • 12.1 What Is the Evidence that Genes Code
      for Proteins?
    • 12.2 How Does Information Flow from Genes
      to Proteins?
    • 12.3 How Is the Information Content in DNA
      Transcribed to Produce RNA?
    • 12.4 How Is RNA Translated into Proteins?
    • 12.5 What Happens to Polypeptides after
      Translation?
    • 12.6 What Are Mutations?
12.1 What Is the Evidence that Genes Code for Proteins?


    The molecular basis of phenotypes was
     known before it was known that DNA is
     the genetic material.
    Studies of many different organisms
     showed that major phenotypic
     differences were due to specific
     proteins.
12.1 What Is the Evidence that Genes Code for Proteins?


   Model organisms: easy to grow or
    observe; show the phenomenon to be
    studied
   Assume that results from one organism
    can be applied to others
   Examples: pea plants, Drosophila, E. coli,
    common bread mold Neurospora crassa
12.1 What Is the Evidence that Genes Code for Proteins?


    Neurospora is haploid for most of its life
     cycle.
    Wild-type strains have enzymes to
     catalyze all reactions needed to make
     cell constituents—prototrophs.
    Beadle and Tatum used X-rays as
     mutagens. Mutants were auxotrophs—
     needed additional nutrients to grow.
12.1 What Is the Evidence that Genes Code for Proteins?


   For each auxotrophic strain, they found a
    single compound that would support
    growth of that strain.
   Suggested the one-gene, one-enzyme
    hypothesis
Figure 12.1 One Gene, One Enzyme (Part 1)
Figure 12.1 One Gene, One Enzyme (Part 2)
12.1 What Is the Evidence that Genes Code for Proteins?


   Beadle and Tatum found several different
    arg mutant strains—had to be supplied
    with arginine.
   arg mutants could have mutations in the
    same gene; or in different genes that
    governed steps of a biosynthetic
    pathway.
12.1 What Is the Evidence that Genes Code for Proteins?


   arg mutants were grown in the presence
    of compounds suspected to be
    intermediates in the biosynthetic
    pathway for arginine.
   This confirmed that each mutant was
    missing a single enzyme in the
    pathway.
12.1 What Is the Evidence that Genes Code for Proteins?


  The gene-enzyme relationship has been
   revised to the one-gene, one-polypeptide
   relationship.
  Example: In hemoglobin, each polypeptide
   chain is specified by a separate gene.
  Other genes code for RNA that is not
   translated to polypeptides; some genes are
   involved in controlling other genes.
12.2 How Does Information Flow from Genes to Proteins?


    Expression of a gene to form a
     polypeptide:
    • Transcription—copies information from
      gene to a sequence of RNA.
    • Translation—converts RNA sequence to
      amino acid sequence.
12.2 How Does Information Flow from Genes to Proteins?


  RNA, ribonucleic acid differs from DNA:
  • Usually one strand
  • The sugar is ribose
  • Contains uracil (U) instead of thymine (T)
12.2 How Does Information Flow from Genes to Proteins?


    RNA can pair with a single strand of
     DNA, except that adenine pairs with
     uracil instead of thymine.
    Single-strand RNA can fold into complex
     shapes by internal base pairing.
Figure 12.2 The Central Dogma




       The central dogma of molecular biology:
    information flows in one direction when genes
            are expressed (Francis Crick).
12.2 How Does Information Flow from Genes to Proteins?


    The central dogma raised two questions:
    • How does genetic information get from
      the nucleus to the cytoplasm?
    • What is the relationship between a DNA
      sequence and an amino acid sequence?
12.2 How Does Information Flow from Genes to Proteins?


    Messenger hypothesis—messenger
     RNA (mRNA) forms as a
     complementary copy of DNA and
     carries information to the cytoplasm.
    This process is transcription.
Figure 12.3 From Gene to Protein
12.2 How Does Information Flow from Genes to Proteins?


    Adapter hypothesis—an adapter
     molecule that can bind amino acids, and
     recognize a nucleotide sequence—
     transfer RNA (tRNA).
    tRNA molecules carrying amino acids
     line up on mRNA in proper sequence for
     the polypeptide chain—translation.
12.2 How Does Information Flow from Genes to Proteins?

 Exception to the central dogma:
 Viruses: acellular particles that reproduce
  inside cells; many have RNA instead of DNA.
12.2 How Does Information Flow from Genes to Proteins?


    Synthesis of DNA from RNA is reverse
     transcription.
    Viruses that do this are retroviruses.
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?


    Within each gene, only one strand of
     DNA is transcribed—the template
     strand.
    Transcription produces mRNA; the same
     process is used to produce tRNA and
     rRNA.
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?


    RNA polymerases catalyze synthesis of
     RNA.
    RNA polymerases are processive—a
     single enzyme-template binding results
     in polymerization of hundreds of RNA
     bases.
Figure 12.4 RNA Polymerase
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?


    Transcription occurs in three phases:
    • Initiation
    • Elongation
    • Termination
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?


    Initiation requires a promoter—a
     special sequence of DNA.
    RNA polymerase binds to the promoter.
    Promoter tells RNA polymerase where to
     start, which direction to go in, and which
     strand of DNA to transcribe.
    Part of each promoter is the initiation
     site.
Figure 12.5 DNA Is Transcribed to Form RNA (A)
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?


    Elongation: RNA polymerase unwinds
     DNA about 10 base pairs at a time;
     reads template in 3′ to 5′ direction.
    The RNA transcript is antiparallel to the
     DNA template strand.
    RNA polymerases do not proofread and
     correct mistakes.
Figure 12.5 DNA Is Transcribed to Form RNA (B)
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?


    Termination: specified by a specific DNA
     base sequence.
    Mechanisms of termination are complex
     and varied.
    Eukaryotes—first product is a pre-mRNA
     that is longer than the final mRNA and
     must undergo processing.
Figure 12.5 DNA Is Transcribed to Form RNA (C)
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?


    The genetic code: specifies which amino
     acids will be used to build a protein
    Codon: a sequence of three bases. Each
     codon specifies a particular amino acid.
    Start codon: AUG—initiation signal for
     translation
    Stop codons: stops translation and
     polypeptide is released
Figure 12.6 The Genetic Code
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?


    For most amino acids, there is more than
     one codon; the genetic code is
     redundant.
    But not ambiguous—each codon
     specifies only one amino acid.
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?


    The genetic code is nearly universal: the
     codons that specify amino acids are the
     same in all organisms.
    Exceptions: within mitochondria and
     chloroplasts, and in one group of
     protists.
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?


    This common genetic code is a common
     language for evolution.
    The code is ancient and has remained
     intact throughout evolution.
    The common code also facilitates genetic
     engineering.
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?


    How was the code deciphered?
    20 “code words” (amino acids) are written
     with only four “letters.”
    Triplet code seemed likely: could account
     for 4 × 4 × 4 = 64 codons.
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?


    Nirenberg and Matthaei used artificial
     polynucleotides instead of mRNA as a
     messenger.
    Then they identified the polypeptide that
     resulted.
Figure 12.7 Deciphering the Genetic Code
12.4 How Is RNA Translated into Proteins?


    tRNA, the adaptor molecule: for each
     amino acid, there is a specific type or
     “species” of tRNA.

    Functions of tRNA:
    • Carries an amino acid
    • Associates with mRNA molecules
    • Interacts with ribosomes
Figure 12.8 Transfer RNA
12.4 How Is RNA Translated into Proteins?


    The conformation (three-dimensional
     shape) of tRNA results from base
     pairing (H bonds) within the molecule.
    3′ end is the amino acid attachment
     site—binds covalently. Always CCA.
    Anticodon: site of base pairing with
     mRNA. Unique for each species of
     tRNA.
12.4 How Is RNA Translated into Proteins?


    Example:
    DNA codon for arginine: 3′-GCC-5′
    Complementary mRNA: 3′-CGG-5′
    Anticodon on the tRNA: 3′-GCC-5′ This
     tRNA is charged with arginine.
12.4 How Is RNA Translated into Proteins?

    Wobble: specificity for the base at the 3′
     end of the codon is not always
     observed.
    Example: codons for alanine—GCA,
     GCC, and GCU—are recognized by the
     same tRNA.
    Allows cells to produce fewer tRNA
     species; but not in all cases—the
     genetic code remains unambiguous.
12.4 How Is RNA Translated into Proteins?


    Charging a tRNA with the correct amino
     acid—amino-acyl-tRNA synthetases.
    Each enzyme is specific for one amino
     acid and its corresponding tRNA.
    The enzymes have three-part active
     sites: they bind a specific amino acid, a
     specific tRNA, and ATP.
Figure 12.9 Charging a tRNA Molecule (Part 1)
Figure 12.9 Charging a tRNA Molecule (Part 2)
12.4 How Is RNA Translated into Proteins?


    The activating enzymes are highly
     specific.
    Amino acid is attached to the 3′ end of
     tRNA by an energy-rich bond—this will
     provide energy for synthesis of the
     peptide bond to join amino acids.
12.4 How Is RNA Translated into Proteins?


    Experiment by Benzer and others:
    Chemically changed cysteine already
     bound to tRNA to alanine.
    Resulting polypeptide had alanine in
     every place that cysteine should be.
    Protein synthesis machinery recognizes
     the anticodon, not the amino acid.
12.4 How Is RNA Translated into Proteins?


    Ribosome: the workbench—holds
     mRNA and tRNA in the correct positions
     to allow assembly of polypeptide chain.
    Ribosomes are not specific, they can
     make any type of protein.
12.4 How Is RNA Translated into Proteins?


    Ribosomes have two subunits, large and
     small.
    In eukaryotes, the large subunit has three
      molecules of ribosomal RNA (rRNA)
      and 45 different proteins in a precise
      pattern.
    The small subunit has one rRNA and 33
     proteins.
12.4 How Is RNA Translated into Proteins?


    Subunits are held together by ionic and
     hydrophobic forces (not covalent
     bonds).
    When not active in translation, the
     subunits exist separately.
Figure 12.10 Ribosome Structure
12.4 How Is RNA Translated into Proteins?


    Large subunit has three tRNA binding
     sites:
    • A site binds with anticodon of charged
      tRNA.
    • P site is where tRNA adds its amino
      acid to the growing chain.
    • E site is where tRNA sits before being
      released.
12.4 How Is RNA Translated into Proteins?


    Hydrogen bonds form between the
     anticodon of tRNA and the codon of
     mRNA.
    Small subunit rRNA validates the
     match—if hydrogen bonds have not
     formed between all three base pairs, it
     must be an incorrect match, and the
     tRNA is rejected.
12.4 How Is RNA Translated into Proteins?


    Translation also occurs in three steps:
    • Initiation
    • Elongation
    • Termination
12.4 How Is RNA Translated into Proteins?


    Initiation:
    An initiation complex forms—charged
     tRNA and small ribosomal subunit, both
     bound to mRNA.
    rRNA binds to recognition site on
     mRNA—the Shine-Dalgarno sequence,
     “upstream” from the start codon.
Figure 12.11 The Initiation of Translation (Part 1)
Figure 12.11 The Initiation of Translation (Part 2)
12.4 How Is RNA Translated into Proteins?


    Start codon is AUG; first amino acid is
     always methionine, which may be
     removed after translation.
    The large subunit joins the complex, the
     charged tRNA is now in the P site of the
     large subunit.
    Initiation factors are responsible for
      assembly of the initiation complex.
12.4 How Is RNA Translated into Proteins?


    Elongation: the second charged tRNA
     enters the A site.
    Large subunit catalyzes two reactions:
    • Breaks bond between tRNA in P site
      and its amino acid.
    • Peptide bond forms between that amino
      acid and the amino acid on tRNA in the
      A site.
Figure 12.12 The Elongation of Translation (Part 1)
Figure 12.12 The Elongation of Translation (Part 2)
12.4 How Is RNA Translated into Proteins?


    The large subunit has peptidyl
     transferase activity.
    RNA acts as the catalyst; normally
     proteins are catalysts.
    Supports the idea that catalytic RNA
     evolved before DNA.
12.4 How Is RNA Translated into Proteins?


    When the first tRNA has released its
     methionine, it moves to the E site and
     dissociates from the ribosome—can
     then become charged again.
    Elongation occurs as the steps are
     repeated, assisted by proteins called
     elongation factors.
12.4 How Is RNA Translated into Proteins?


    Termination: translation ends when a
     stop codon enters the A site.
    Stop codon binds a protein release
     factor—allows hydrolysis of bond
     between polypeptide chain and tRNA on
     the P site.
    Polypeptide chain—C terminus is the last
     amino acid added.
Figure 12.13 The Termination of Translation (Part 1)
Figure 12.13 The Termination of Translation (Part 2)
Figure 12.13 The Termination of Translation (Part 3)
Table 12.1
12.4 How Is RNA Translated into Proteins?


    Several ribosomes can work together to
     translate the same mRNA, producing
     multiple copies of the polypeptide.
    A strand of mRNA with associated
     ribosomes is called a polyribosome or
     polysome.
Figure 12.14 A Polysome (Part 1)
Figure 12.14 A Polysome (Part 2)
12.5 What Happens to Polypeptides after Translation?


    Posttranslational aspects of protein
     synthesis:
    Polypeptide may be moved from
     synthesis site to an organelle, or out of
     the cell.
    Polypeptides are often modified with
     more chemical groups.
12.5 What Happens to Polypeptides after Translation?


    Polypeptide folds as it emerges from the
     ribosome.
    The amino acid sequence determines the
     pattern of folding.
    Amino acid sequence also contains a
     signal sequence—an “address label.”
12.5 What Happens to Polypeptides after Translation?


    Amino acid sequence gives a set of
     instructions:
    “Finish translation and send to an
     organelle.”
           OR
    “Stop translation, go to the ER, finish
     synthesis there.”
Figure 12.15 Destinations for Newly Translated Polypeptides in a Eukaryotic Cell
12.5 What Happens to Polypeptides after Translation?


   Conformation of signal sequences allow
    them to bind specific receptor proteins—
    docking proteins—on outer membranes
    of organelles.
   Receptor forms a channel that the protein
    passes through. May be unfolded at this
    time by chaperonins.
12.5 What Happens to Polypeptides after Translation?

    If the protein is sent to the ER:
    • Signal sequence binds to a signal
      receptor particle, before translation is
      done.
    • Ribosome attaches to a receptor on the
      ER, the growing polypeptide chain
      passes through the channel.
    • An enzyme removes the signal
      sequence.
Figure 12.16 A Signal Sequence Moves a Polypeptide into the ER (Part 1)
Figure 12.16 A Signal Sequence Moves a Polypeptide into the ER (Part 2)
12.5 What Happens to Polypeptides after Translation?


    Sugars may be added in the Golgi
     apparatus—the resulting glycoproteins
     end up in the plasma membrane,
     lysosomes, or vacuoles.
12.5 What Happens to Polypeptides after Translation?


   Protein modifications:
   Proteolysis: cutting the polypeptide chain,
    by proteases.
   Glycosylation: addition of sugars to form
    glycoproteins.
   Phosphorylation: addition of phosphate
    groups by kinases. Charged phosphate
    groups change the conformation.
Figure 12.17 Posttranslational Modifications of Proteins
12.6 What Are Mutations?


    Somatic mutations occur in somatic
     (body) cells. Mutation is passed to
     daughter cells, but not to sexually
     produced offspring.
    Germ line mutations occur in cells that
     produce gametes. Can be passed to
     next generation.
12.6 What Are Mutations?


    Conditional mutants: express
     phenotype only under restrictive
     conditions.
    Example: the allele may code for an
     enzyme that is unstable at certain
     temperatures.
12.6 What Are Mutations?


    All mutations are alterations of the
     nucleotide sequence.
    Point mutations: change in a single
     base pair—loss, gain, or substitution of
     a base.
    Chromosomal mutations: change in
     segments of DNA—loss, duplication, or
     rearrangement.
12.6 What Are Mutations?

    Point mutations can result from
     replication and proofreading errors, or
     from environmental mutagens.
    Silent mutations have no effect on the
     protein because of the redundancy of
     the genetic code.
    Silent mutations result in genetic diversity
     not expressed as phenotype
     differences.
12.6 What Are Mutations?
12.6 What Are Mutations?

    Missense mutations: base substitution
     results in amino acid substitution.
12.6 What Are Mutations?


    Sickle allele for human β-globin is a
     missense mutation.
    Sickle allele differs from normal by only
     one base—the polypeptide differs by
     only one amino acid.
    Individuals that are homozygous have
      sickle-cell disease.
Figure 12.18 Sickled and Normal Red Blood Cells
12.6 What Are Mutations?


    Nonsense mutations: base substitution
     results in a stop codon.
12.6 What Are Mutations?

    Frame-shift mutations: single bases
     inserted or deleted—usually leads to
     nonfunctional proteins.
12.6 What Are Mutations?

    Chromosomal mutations:
    Deletions—severe consequences unless
     it affects unnecessary genes or is
     masked by normal alleles.
    Duplications—if homologous
     chromosomes break in different places
     and recombine with the wrong partners.
Figure 12.19 Chromosomal Mutations (A, B)
12.6 What Are Mutations?

    Chromosomal mutations:
    Inversions—breaking and rejoining, but
     segment is “flipped.”
    Translocations—segment of DNA
     breaks off and is inserted into another
     chromosome. Can cause duplications
     and deletions. Meiosis can be
     prevented if chromosome pairing is
     impossible.
Figure 12.19 Chromosomal Mutations (C, D)
12.6 What Are Mutations?

    Spontaneous mutations—occur with no
     outside influence. Several mechanisms:
    • Bases can form tautomers—different
      forms; rare tautomer can pair with the
      wrong base.
    • Chemical reactions may change bases
      (e.g., loss of amino group).
12.6 What Are Mutations?

    • Replication errors—some escape
      detection and repair.
    • Nondisjunction in meiosis.
12.6 What Are Mutations?

    Induced mutation—due to an outside
     agent, a mutagen.
    Chemicals can alter bases (e.g., nitrous
     acid can cause deamination).
    Some chemicals add other groups to
     bases (e.g., benzpyrene adds a group
     to guanine and prevents base pairing).
     DNA polymerase will then add any base
     there.
12.6 What Are Mutations?

    Ionizing radiation such as X-rays create
      free radicals—highly reactive—can
      change bases, break sugar phosphate
      bonds.
    UV radiation is absorbed by thymine,
     causing it to form covalent bonds with
     adjacent nucleotides—disrupts DNA
     replication.
Figure 12.20 Spontaneous and Induced Mutations (Part 1)
Figure 12.20 Spontaneous and Induced Mutations (Part 2)
12.6 What Are Mutations?

    Mutation provides the raw material for
     evolution in the form of genetic diversity.
    Mutations can harm the organism, or be
     neutral.
    Occasionally, a mutation can improve an
     organism’s adaptation to its
     environment, or become favorable as
     conditions change.
12.6 What Are Mutations?

   Complex organisms tend to have more
    genes than simple organisms.
   If whole genes are duplicated, the new
     genes would be surplus genetic
     information.
   Extra copies could lead to the production of
    new proteins.
   New genes can also arise from
    transposable elements (see Chapters 13
    and 14).

				
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