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					 Chapter 17
From Gene to
   Protein
          Question?
u How does DNA control a cell?
u By controlling Protein
  Synthesis.
u Proteins are the link between
  genotype and phenotype.
           For tests:
u Name(s) of experimenters
u Outline of the experiment
u Result of the experiment and
  its importance
    1909 - Archibald Garrod
u Suggested genes control
  enzymes that catalyze
  chemical processes in cells.
u Inherited Diseases - “inborn
  errors of metabolism” where
  a person can’t make an
  enzyme.
            Example
u Alkaptonuria - where urine
  turns black after exposure to
  air.
u Lacks - an enzyme to
  metabolize alkapton.
          George Beadle and
            Edward Tatum
u   Worked with Neurospora and
    proved the link between
    genes and enzymes.


    Neurospora
    Pink bread mold
         Experiment
u Grew Neurospora on agar.
u Varied the nutrients.
u Looked for mutants that
  failed to grow on minimum
  agar.
            Results
u Three classes of mutants for
  Arginine Synthesis.
u Each mutant had a different
  block in the Arginine
  Synthesis pathway.
         Conclusion
u Mutations were abnormal
  genes.
u Each gene dictated the
  synthesis of one enzyme.
u One Gene - One Enzyme
  Hypothesis.
       Current Hypothesis
u   One Gene - One Polypeptide
    Hypothesis (because of 4th
    degree structure).
       Central Dogma
                DNA
Transcription


                RNA
 Translation


          Polypeptide
          Explanation
u DNA - the Genetic code or
  genotype.
u RNA - the message or
  instructions.
u Polypeptide - the product for
  the phenotype.
        Genetic Code
u Sequence of DNA bases that
  describe which Amino Acid
  to place in what order in a
  polypeptide.
u The genetic code gives the
  primary protein structure.
          Code Basis
If you use:
u 1 base = 1 amino acid
u 4 bases = 4 amino acids
u 41 = 4 combinations, which
  are not enough for 20 AAs.
          If you use:
u 2 bases = 1 amino acid
u Ex – AT, TA, CA, GC
   2
u 4 = 16 amino acids
u Still not enough combinations.
          If you use:
u 3 bases = 1AA
u Ex – CAT, AGC, TTT
   3
u 4 = 64 combinations
u More than enough for 20
  amino acids.
          Genetic Code
u Is based on triplets of bases.
u Has redundancy; some AA's
  have more than 1 code.
u Proof - make artificial RNA and
  see what AAs are used in
  protein synthesis (early 1960’s).
             Codon
u A 3-nucleotide “word” in the
  Genetic Code.
u 64 possible codons known.
        DNA vs RNA
       DNA           RNA
Sugar – deoxyribose ribose
Bases – ATGC        AUGC
Backbones – 2       1
Size – very large   small
Use – genetic code  varied
      Codon Dictionary
u Start- AUG (Met)
u Stop- UAA
         UAG
         UGA
u 60 codons for the other 19
  AAs.
         For Testing:
u Be able to “read” a DNA or
  RNA message and give the
  AA sequence.
u RNA Genetic Code Table will
  be provided.
      Code Redundancy
u Third base in a codon shows
  "wobble”.
u First two bases are the most
  important in reading the code
  and giving the correct AA.
  The third base often doesn’t
  matter.
       Code Evolution
u The genetic code is nearly
  universal.
u Ex: CCG = proline (all life)
u Reason - The code must have
  evolved very early. Life on
  earth must share a common
  ancestor.
         Reading Frame and
            Frame Shift
u   The “reading” of the code is every
    three bases (Reading Frame)
u   Ex: the red cat ate the rat
u   Frame shift – improper groupings
    of the bases
u   Ex: thr edc ata tat her at
u   The “words” only make sense if
    “read” in this grouping of three.
          Transcription
u   Process of making RNA from
    a DNA template.
       Transcription Steps
1.   RNA Polymerase Binding
2.   Initiation
3.   Elongation
4.   Termination
         RNA Polymerase
u   Enzyme for building RNA
    from RNA nucleotides.
              Binding
u   Requires that the enzyme find
    the “proper” place on the
    DNA to attach and start
    transcription.
            Binding
u Is a complicated process
u Uses Promoter Regions on
  the DNA (upstream from the
  information for the protein)
u Requires proteins called
  Transcription Factors.
           TATA Box
u Short segment of T,A,T,A
u Located 25 nucleotides
  upstream for the initiation
  site.
u Recognition site for
  transcription factors to bind
  to the DNA.
    Transcription Factors
u Proteins that bind to DNA
  before RNA Polymerase.
u Recognizes TATA box,
  attaches, and “flags” the spot
  for RNA Polymerase.
     Transcription Initiation
           Complex
u   The complete assembly of
    transcription factors and RNA
    Polymerase bound to the
    promoter area of the DNA to
    be transcribed.
            Initiation
u Actual unwinding of DNA to
  start RNA synthesis.
u Requires Initiation Factors.
          Elongation
u RNA Polymerase untwists
  DNA 1 turn at a time.
u Exposes 10 DNA bases for
  pairing with RNA nucleotides.
          Elongation
u Enzyme moves 5’      3’.
u Rate is about 60 nucleotides
  per second.
           Comment
u Each gene can be read by
  sequential RNA Polymerases
  giving several copies of RNA.
u Result - several copies of the
  protein can be made.
         Termination
u DNA sequence that tells RNA
  Polymerase to stop.
u Ex: AATAAA
u RNA Polymerase detaches
  from DNA after closing the
  helix.
         Final Product
u Pre-mRNA
u This is a “raw” RNA that will
  need processing.
    Modifications of RNA
1. 5’ Cap
2. Poly-A Tail
3. Splicing
            5' Cap
u Modified Guanine nucleotide
  added to the 5' end.
u Protects mRNA from
  digestive enzymes.
u Recognition sign for
  ribosome attachment.
          Poly-A Tail
u 150-200 Adenine nucleotides
  added to the 3' tail
u Protects mRNA from
  digestive enzymes.
u Aids in mRNA transport from
  nucleus.
           Comment
u The head and tail areas often
  contain “leaders” and
  “trailers”, areas of RNA that
  are not read.
u Similar to leaders or trailers
  on cassette tapes.
        RNA Splicing
u Removal of non-protein
  coding regions of RNA.
u Coding regions are then
  spliced back together.
            Introns
u Intervening sequences.
u Removed from RNA.
            Exons
u Expressed sequences of
  RNA.
u Translated into AAs.
        Spliceosome
u Cut out Introns and join
  Exons together.
u Made of snRNA and snRNP.
             snRNA
u Small Nuclear RNA.
u 150 nucleotides long.
u Structural part of
  spliceosomes.
           snRNPs
u ("snurps")
u Small Nuclear
  Ribonucleoprotiens
u Made of snRNA and proteins.
u Join with other proteins to
  form a spliceosome.
          Ribozymes
u RNA molecules that act as
  enzymes.
u Are sometimes Intron RNA
  and cause splicing without a
  spliceosome.
      Introns - Function
u Left-over DNA (?)
u Way to lengthen genetic
  message.
u Old virus inserts (?)
u Way to create new proteins.
Final RNA Transcript
            Translation
u   Process by which a cell
    interprets a genetic message
    and builds a polypeptide.
     Materials Required
u tRNA
u Ribosomes
u mRNA
    Transfer RNA = tRNA
u Made by transcription.
u About 80 nucleotides long.
u Carries AA for polypeptide
  synthesis.
      Structure of tRNA
u Has double stranded regions
  and 3 loops.
u AA attachment site at the 3'
  end.
u 1 loop serves as the
  Anticodon.
          Anticodon
u Region of tRNA that base
  pairs to mRNA codon.
u Usually is a compliment to
  the mRNA bases, so reads
  the same as the DNA codon.
           Example
u DNA - GAC
u mRNA - CUG
u tRNA anticodon - GAC
            Comment
u "Wobble" effect allows for 45
  types of tRNA instead of 61.
u Reason - in the third position,
  U can pair with A or G.
u Inosine (I), a modified base in
  the third position can pair
  with U, C, or A.
         Importance
u Allows for fewer types of
  tRNA.
u Allows some mistakes to
  code for the same AA which
  gives exactly the same
  polypeptide.
       Aminoacyl-tRNA
        Synthetases
u Family of Enzymes.
u Add AAs to tRNAs.
u Active site fits 1AA and 1
  type of tRNA.
u Uses a “secondary genetic”
  code to load the correct AA
  to each tRNA.
         Ribosomes
u Two subunits made in the
  nucleolus.
u Made of rRNA (60%)and
  protein (40%).
u rRNA is the most abundant
  type of RNA in a cell.
           Large subunit
Proteins




rRNA
Both sununits
           Large Subunit
u   Has 3 sites for tRNA.
u   P site: Peptidyl-tRNA site -
    carries the growing polypeptide
    chain.
u   A site: Aminoacyl-tRNA site -
    holds the tRNA carrying the next
    AA to be added.
u   E site: Exit site
      Translation Steps
1. Initiation
2. Elongation
3. Termination
           Initiation
u Brings together:
u mRNA
u A tRNA carrying the 1st AA
u 2 subunits of the ribosome
       Initiation Steps:
1. Small subunit binds to the
     mRNA.
2. Initiator tRNA (Met, AUG)
     binds to mRNA.
3. Large subunit binds to
     mRNA. Initiator tRNA is in
     the P-site
           Initiation
u Requires other proteins
  called "Initiation Factors”.
u GTP used as energy source.
     Elongation Steps:
1. Codon Recognition
2. Peptide Bond Formation
3. Translocation
       Codon Recognition
u   tRNA anticodon matched to
    mRNA codon in the A site.
        Peptide Bond
         Formation
u A peptide bond is formed
  between the new AA and the
  polypeptide chain in the  P
  -site.
u Bond formation is by rRNA
  acting as a ribozyme
       After bond formation
u   The polypeptide is now
    transferred from the tRNA in
    the P-site to the tRNA in the A
    -site.
         Translocation
u tRNA in P-site is released.
u Ribosome advances 1 codon,
  5’ 3’.
u tRNA in A-site is now in the P
  -site.
u Process repeats with the next
  codon.
             Comment
u   Elongation takes 60
    milliseconds for each AA
    added.
         Termination
u Triggered by stop codons.
u Release factor binds in the
  A-site instead of a tRNA.
u H2O is added instead of AA,
  freeing the polypeptide.
u Ribosome separates.
       Polyribosomes
u Cluster of ribosomes all
  reading the same mRNA.
u Another way to make multiple
  copies of a protein.
AP Bio




         Carol - Barr
         -Reeve
          Homework
u Read – Chapter 17, 42
u Lab – Chapter 42 – due next
  lab period
u Chapter 17 – Wed. 2/2
Prokaryotes
           Homework
u Read Chapter 17, 18
u Bacteria transformation – due
  next lab period
u Lab this week – DNA
  sequencing and other stuff
u Chapter 17 – Mon. Feb. 1
u Take home exam – due Fri. 1/29
             Comment
u   Polypeptide usually needs to
    be modified before it
    becomes functional.
           Examples
u Sugars, lipids, phosphate
  groups added.
u Some AAs removed.
u Protein may be cleaved.
u Join polypeptides together
  (Quaternary Structure).
      Signal Hypothesis
u “Clue” on the growing
  polypeptide that causes
  ribosome to attach to ER.
u All ribosomes are “free”
  ribosomes unless clued by
  the polypeptide to attach to
  the ER.
               Result
u Protein is made directly into
  the ER .
u Protein targeted to desired
  location (e.g. secreted protein).
u “Clue” (the first 20 AAs are
  removed by processing).
           Mutations
u Changes in the genetic
  makeup of a cell.
u May be at chromosome
  (review chapter 15) or DNA
  level
    DNA or Point Mutations
u Changes in one or a few
  nucleotides in the genetic
  code.
u Effects - none to fatal.
        Types of Point
          Mutations
1. Base-Pair Substitutions
2. Insertions
3. Deletions
      Base-Pair Substitution
u   The replacement of 1 pair of
    nucleotides by another pair.
Sickle Cell Anemia
   Types of Substitutions
1. Missense - altered codons,
 still code for AAs but not the
 right ones
2. Nonsense - changed codon
 becomes a stop codon.
          Question?
u What will the "Wobble" Effect
  have on Missense?
u If the 3rd base is changed,
  the AA may still be the same
  and the mutation is “silent”.
          Comment
u Silent mutations may still
  have an effect by slowing
  down the “speed” of making
  the protein.
u Reason – harder to find some
  tRNAs than others.
        Missense Effect
u Can be none to fatal
  depending on where the AA
  was in the protein.
u Ex: if in an active site - major
  effect. If in another part of
  the enzyme - no effect.
       Nonsense Effect
u Stops protein synthesis.
u Leads to nonfunctional
  proteins unless the mutation
  was near the very end of the
  polypeptide.
      Sense Mutations
u The changing of a stop codon
  to a reading codon.
u Result - longer polypeptides
  which may not be functional.
u Ex. “heavy” hemoglobin
    Insertions & Deletions
u The addition or loss of a base
  in the DNA.
u Cause frame shifts and
  extensive missense,
  nonsense or sense
  mutations.
           Question?
u Loss of 3 nucleotides is often
  not a problem.
u Why?
u Because the loss of a 3 bases
  or one codon restores the
  reading frame and the protein
  may still be able to function.
           Mutagenesis
u   Process of causing mutations
    or changes in the DNA.
              Mutagens
uMaterials that cause DNA
 changes.
1. Radiation
    ex: UV light, X-rays
2. Chemicals
    ex: 5-bromouracil
          Spontaneous
           Mutations
u   Random errors during DNA
    replication.
              Comment
u   Any material that can
    chemically bond to DNA,
    or is chemically similar to the
    nitrogen bases, will often be
    a very strong mutagen.
       What is a gene?
u A gene is a region of DNA
  that can be expressed to
  produce a final functional
  product.
u The product can be a protein
  or a RNA molecule
        Protein vs RNA
u Protein – usually structure or
  enzyme for phenotype
u RNA – often a regulatory
  molecule which will be
  discussed in future chapters.
           Summary
u Know Beadle and Tatum.
u Know the central dogma.
u Be able to “read” the genetic
  code.
u Be able to describe the
  events of transcription and
  translation.
           Summary
u Be able to discuss RNA and
  protein processing.
u Be able to describe and
  discuss mutations.
u Be able to discuss “what is a
  gene”.

				
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