Unit 5A: Molecular Genetics

1

• Unit 5A Molecular Genetics
• Section 1 History of DNA and its Structure

2
A Brief History of Science

• The discovery of Mendels work brought many
  questions.
• Scientists knew
• The genetic material must be able to replicate
  itself
• Must be able to control living processes
• Most biologists thought until 1940s that
  proteins were the information molecules
• Very complex
• Much variety

3
DNA Early Evidence for Importance

• 1869 Friedreich Meischer (Swiss)
• Bandages from patients in hospitals
• Isolated a phosphate-containing acid from the
  nucleus Nuclein
• 1919 Robert Feulgen
• Basic fuchsin turns purple in presence of DNA.

4
Griffith Transformation

• 1928 Fredrick Griffith Killed in 1941 in the
  Blitz in London.
• Two strains of bacteria, back then called
  Diplococcus (now Streptococcus) pneumoniae make
  two types of colonies
• Smooth and round (S) strain - Virulent / lethal
• Had a protective coat around the bacteria
• Rough (R) Nonvirulent
• Did not have protective coat around bacteria and
  therefore immune system could fight

5
Griffith Transformation - 1928

• Griffith sought a pneumonia vaccine (none exist
  today)
• If S was injected, mice died.
• If R injected, mice lived.
• If heat-treat S, then inject, mice lived.
• If combine heat-treated S with R, and inject,
  mice died!
• This was the question this should not have
  occurred.
• Live S bacteria can be isolated from the dead
  mice, but it was heated and should not have been
  found.
• Griffin determine that transformation had
  occurred.

Transformation had occurred the R bacteria took
something up from the dead S a transforming
principle (molecule) of some sort could changed
the R bacteria!
6
Another Key Experiment - 1930

• Max Delbruck, Alfred Hershey, and Salvador Luria
• Bacteria could be attacked by a virus
  (bacteriophage)
• Within 60 seconds, the bacteria started to make a
  new protein the capsid (outer coat) of the virus
• The transforming principle at work!

7
Avery Experiment DNA or Proteins - 1944

• In 1944, O.T. Avery, C.M. MacLeod and M. McCarty
  who were working at the Rockefeller Institute
  decided to repeat Griffiths experiments.
• Their goal was to control all the variables as
  closely as they possible could.
• They decided the genetic material was either
  Nucleic Acids or Proteins but they were not sure
  which one.
• Avery (lead scientist) said he felt like it would
  be protein because there are so many amino acids
  that could be rearranged for genetic information
  as compared to the five types of nucleic acids.
• (Literature today considers this the first
  experiment of the transforming principal and
  nucleic acids).

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Edwin Chargaff

• 1930/40's Chargaffs Rules
• Chemist hard-working
• Discovered
• The molar quantity of A molar quantity of T
• The molar quantity of C molar quantity of G
• He did not know what this meant at the time and
  was in the race to determine the structure of
  DNA.
• We now know that each species has its own
  peculiar ATGC complement, but always, AT CG

9
Hershey and Chase - 1952

• Alfred Hershey had been working with
  bacteriophages since 1930.
• Martha Chase (female) was one of the first women
  in the field of genetics who joined him as his
  research assistant.

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Hershey-Chase

• Labeled virus capsid (like a cell wall) with 35S
  to the protein which is found on the outside of
  the virus.
• They attached 32P to the nucleic acids inside the
  capsid.
• Remember S is in Proteins
• Remember P is in Nucleic Acids
• Allowed the bacteriophage to attach to bacteria
  and infect the organism.
• They used a blender to break up the bacterial
  cells.
• They used the method of centrifugation to
  separate the bacteria and phage.
• They found the sulfur in the supernantant and the
  phosphorous still in the bacteria.
• Therefore they concluded that nucleic acids was
  the genetic material.

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Hershey Chase Experiment - Visual
12
Hershey Chase Experiment - Video

• http//highered.mcgraw-hill.com/sites/0072437316/s
  tudent_view0/chapter14/animations.html

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The Race to discovery the Structure of DNA

• Remember that all of this was going on at one
  time in different locations in the world. Some
  of the people were in England, US and France and
  since Hershey and Chases experiment there was a
  race to discover the structure of DNA.
• The scientist that made this discovery was set
• Nobel Prize
• Choose where they wanted to work.
• Fame Fortune in speaking engagements.

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Players in the Game

• OT Avery Pic B-4
• Lawrence Bragg
• Edwin Chargaff Pic B-4
• Francis Crick
• Rosalind Franklin
• Linus Pauling
• James Watson
• Maurice Wilkins

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X-Ray Diffraction

• 1940-1953 Maurice Wilkins (New Zealand) and
  Rosalind Franklin (English) of Kings College,
  London UK X-Ray diffraction analysis of DNA.
• Wilkins and Franklin were already working on this
  project when Crick and Watson entered the
  picture.
• Franklin in Wilkins lab had shown
• Showed
• DNA was a linear molecule
• 3.4 nm repeat along the length
• 0.34 nm repeat along the length, too
• DNA was a helix b/c the diffraction pattern made
  an X
• The photograph that was taken is Photo 51

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Linus Pauling

• Was working on the structure of DNA in the United
  States.
• His son was actually in England as a graduate
  student at the time.
• Watson and Crick were very careful to say things
  around him but they consistently asked about his
  fathers work.
• Some of his work was halted due to Red Scare
  and the pulling of his passport so he was unable
  to attend key conferences in England.

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Watson and Crick

• Crick English phage geneticist at the Cavendish
  labs at Cambridge University, London England
• Watson American doc student in Cricks lab
• Discovery of DNA Structure
• Both visited Wilkins Franklin routinely 1951-53
• Derived the overall concept of the chemical
  relationship
• Considered how Chargaffs rules represented the
  structure of DNA
• Franklins X ray data
• Read Linus Paulings paper on single stranded
  alpha DNA
• Built little tin models of the nucleotides and
  put the DNA model together like a TinkerToy set
• Correctly deduced the structure of DNA
• Published their paper in Nature in 1953

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The Double Helix

• This is the Watson and Crick model worked out in
  1953 and published in a single-page article in
  Nature of that year.
• This form of DNA is called ß-DNA.
• Was convincing structurally gave evidence for
  how DNA replicated (new strand off each of the
  old strands)
• Most famous biology paper ever written!

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Reminder of DNA Structure

• DNA has an inherent polarity
• It is held together by phosphodiester bonds
  between sugar subunits
• Leaves phosphates free at opposite ends.
• Phosphates are attached between the 5' and 3'
  carbons of the sugars
• DNA has 5 and 3 phosphates exposed on each
  strand.
• On the next slide strands are of opposite
  polarity antiparallel strands are hydrogen
  bonded via A pairing with T and C with G they
  are complementary to one another.

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DNA Structure
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Unit 5A Molecular GeneticsSection 2 DNA
Replication
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How Does DNA Replicate?

• Several research groups worked on this question.
  
• 1957 Matthew Meselsohn and Fred Stahl are
  credited with the first real explaination of how
  this occurs.
• Hypotheses
• 1. DNA replication is semiconservative
• One old strand kept with each of the new
  molecules one old paired with one new strand
• 2. DNA replication is conservative
• Double strand maintained intact new strands are
  together in the new molecule
• 3. DNA replication is dispersive
• Strands cut up and the old and new DNA
  interspersed in both new strands

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Hypothesis 1

• In semiconservative replication, each strand
  would give rise to a new strand

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Hypothesis 2

• In conservative replication the old
  double-stranded DNA would be conserved the new
  double-stranded DNA would be entirely new.

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Hypothesis 3

• In dispersive replication, the old strand would
  be cut up, and old pieces interspersed with new
  pieces each strand would have segments of old
  and new double stranded DNA.

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Density Gradient Centrifugation

• Okay, great, we know the structure of DNA now we
  need to understand how it replicates. So the
  idea of density gradient centrifugation was used.
  
• Most common isotope of N has atomic weight of 14
  14N
• A heavy isotope of N has atomic weight of 15 15N
• Bases have lots of N so it would be easy to give
  bacteria either 14N or 15N containing nutrient
  media and see what happens.
• DNA produced with the different isotopes has
  different specific gravity (density)

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Meselsohn and Stahl Growth of cells

• Took advantage of the fact that bacteria take up
  any isotope of N to make DNA
• Used 15N (heavy N) instead of 14N to make
  heavy DNA during a first round of bacterial
  growth
• The 15N containing medium was replaced with 14N
  containing medium in later rounds of growth

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DNA is Harvested and Centrifuged

• The DNA is placed in centrifuge tubes with CsCl
• Subjected to powerful centrifugation CsCl makes
  a density gradient
• The DNA moved to region of same density
• Called isopycnic (same density) centrifugation

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Model Predictions First Growth Cycle

• Grew bacteria continuously in first cycle in
  heavy N 15N -containing medium for many
  generations
• Thus, this DNA would be heavy per unit volume
• Would have high density
• Then grew in 14N for one generation
• Isolated double-stranded DNA from the bacteria
  and subjected to isopycnic CsCl centrifugation
• H1 - If semiconservative, should see medium
  density DNA
• H2 - If replication were conservative, MS should
  see both light and heavy density DNA
• H3 - If dispersive, should see medium density DNA

30
The First Round Eliminated a Conservative
Replication Mechanism

• They observed that the DNA had intermediate
  density

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Second Cycle

• Thus, with one round of growth, conservative
  replication was eliminated
• Bacteria grew through another growth cycle with
  light N medium
• If dispersive, the band would get less denser,
  but denser than DNA produced with only 14N
• If semiconservative, the band would split one of
  medium density, and one of light density

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They Observed Two Bands

• So, DNA must undergo semiconservative replication

33
Meselsohn and Stahl Experiment Video

• http//highered.mcgraw-hill.com/sites/0072437316/s
  tudent_view0/chapter14/animations.html

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Mutations Are Stabilized by Semiconservative
Replication

• The inherent stability and reproducibility of the
  semiconservative mechanism stabilizes any
  mutation that occurs.
• Each strand acts as a template for the other, and
  so the mutation will continue through successive
  generations only in that strand.
• This is a good thing!!!

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DNA Replication
36
DNA Packaging Chromosome Structure

• DNA is greatly wrapped up
• Protects from environment
• Takes up much less space
• Bacterial DNA would be about 1.1-1.5 mm long,
  even though the cell is maybe only 1-3 microns
  long!
• Eukaryotic DNA would be about 1 meter! -
  Eukaryotic DNA which is 100-1000 times more is
  more tightly wrapped
• Histone proteins
• Basic ( charged) negates effect of the (-)
  charged phosphates in DNA

37
DNA Levels of Organization

• Condensed chromosome
• Condensed chromatin 700 nm fiber
• 300 nm looped chromatin
• 30 nm packed nucleosomes
• 11 nm nucleosomes
• 2 nm DNA width

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Nucleosomes

• DNA is wrapped most closely with basic ()
  charged histone proteins
• Histones form nest-like nucleosomes around which
  is wrapped DNA
• DNA linkers hold nucleosomes together
• Nucleosomes are structural, but also aid with
  regulation of DNA replication and expression

39
Bidirectional Replication
Prokaryotic

• DNA replication usually occurs on both sides of
  the origin
• Seen clearly in replicating circular DNA of
  bacteria (a) where there is one origin
• However bidirectional replication is seen in
  eukaryotes too, where there are multiple origins
  of replication (c, d)

Eukaryotic
40
How Does Replication Start?

• The replication complex binds at the origin of
  replication, which is identified by a particular
  base sequence
• DNA Helicase unwinds the DNA and opens the two
  strands, the molecule is held open with
  helix-destabilizing proteins.
• DNA Replication starts in the replication fork.
• Topoisomerases produce breaks in the DNA molecule
  so it does not become knotted, but it will join
  them back as well.

41
Replication Proceeds on Two Strands

• Subunits always add at the 3 end, but the new
  strands elongate in opposite directions so DNA
  grows 5 ? 3
• The leading strand elongates into the fork
• The lagging strand elongates away from the fork
• RNA primers are first laid down in each case
  (red) by a primosome complex (this is so mRNA
  can be made during this time also) it also
  identifies where DNA will have to be ligated
  together.
• Elongation proceeds smoothly on the leading
  strand

42
Leading and Lagging Strands

• As the fork grows, both new strands elongate
  further
• Subunit addition to the 3 end of the lagging
  strand is by 100-2000 base Okazaki fragments.
• The lagging strand grows in a discontinuous
  manner because of the size of the Okazaki
  fragments
• Thats why it lags

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Ligating (connecting) the Lagging Strand pieces

• The RNA primer is degraded between new sections
  of DNA.
• The remaining gap is closed in by DNA ligase

44
Enzymes
45
1, 2, 3 for the Leading Strand

• Helicase unwinds the parent DNA. Topoisomerase,
  helps correct overwinding by opening and closing
  the DNA ahead of the Helicase.
• Proteins stabilize the unwound DNA and hold it
  open.
• RNA Primase is laid down to identify where to
  start mRNA syntheis and nucleic acid addition.
• The leading strand is synthesized continuously by
  adding to the 3 end of the strand by DNA
  Polymerase III.
• DNA Poly II checks the base pairs to make sure
  they are appropriately paired.
• DNA Poly I will remove the primers by breaking
  the hydrogen bonds of the RNA primers so that the
  leading strands can be hooked to the next strand
  of DNA by DNA Ligase.
• The leading strand and lagging strand continue in
  opposite directions in the replication bubble and
  will be connected with the leading strand of
  another portion of the DNA when it reaches the
  back end / start of that strand by DNA Ligase.

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1, 2, 3 for the Lagging Strand

• Helicase unwinds the parent DNA. Topoisomerase,
  helps correct overwinding by opening and closing
  the DNA ahead of the Helicase.
• Proteins stabilize the unwound DNA and hold it
  open.
• RNA Primase is laid down to identify where to
  start mRNA syntheis and nucleic acid addition.
• The lagging strand has various parts called
  Okazaki fragments. Each Okazaki fragment is
  synthesized by adding to the 3 end of the strand
  by DNA Polymerase III along the various fragments
  (usually 4-6 pieces).
• DNA Poly II checks the base pairs to make sure
  they are appropriately paired.
• DNA Poly I will remove the primers by breaking
  the hydrogen bonds of the RNA primers so that the
  leading strands can be hooked to the next strand
  of DNA by DNA Ligase.
• The leading strand and lagging strand continue in
  opposite directions in the replication bubble and
  will be connected with the leading strand of
  another portion of the DNA when it reaches the
  back end / start of that strand by DNA Ligase.

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Videos on DNA Replication

• http//207.207.4.198/pub/flash/24/menu.swf
• http//www.johnkyrk.com/DNAreplication.html

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Cellular Ageing and DNA

• The replication process never entirely completes
  at the ends of the chromosomes
• However, DNA is protected at its ends with long
  strands that do not carry any genetic
  information, called telomeres
• They are restored with a special polymerase
  called telomerase
• Even so, as we age, they become shorter
• They are repaired and lengthened with an enzyme
  called telomerase
• Loss of telomerase activity may be an important
  cause of cellular aging

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Unit 5A Molecular GeneticsSection 3 Gene
Expression
50
Gene Expression

• Cells contain an information system DNA
• Most of the cell is not DNA most of the cell is
  made of protein
• Those components that are not directly protein
  themselves, are controlled or regulated by
  proteins enzymes such as pumps, kinases,
  ATPases, polymerases and motility enzymes, found
  in organelles that make and move cellular
  materials around
• So, the essential key players in the cells
  moment-by-moment existence are proteins.
  Therefore, we must consider . . .
• How is information coded in the information
  system,
• How is that information decoded and interpreted,
  and
• How are proteins produced?
• That is, we must examine gene expression

51
Genes and Enzymes

• The relationship between genotype and phenotype
  was first recognized to affect health and bodily
  function via enzymes
• 1908 Archibald Garrod (English) and Black Urine
  Inborn Errors of Metabolism
• Premise certain diseases arise from metabolic
  disorders
• Tyrosine is broken down through hydroxyphenyl
  pyruvate to homogentisic acid. Normally
  homogentisic acid is passed in the urine
• In the condition called alkaptonuria (or black
  urine) homogentisic acid in the urine oxidizes
  and turns to brown compound upon exposure to air
  interesting but not dangerous condition.
• Garrod proposed that a lost or damaged enzyme was
  responsible for this

52
Alkaptonuria

• Normal tyrosine degradation pathway on the right
• Pathway leading to alkaptonuria is on left such
  individuals lack the enzyme to break down
  homogentisic acid
• The genetic control of protein production is not
  efficiently studied in humans because such
  problems are rare and humans obviously cannot be
  experimentally manipulated

53
Beadle and Tatum

• Established one gene one enzyme concept
• Studied Neurospora bread mold several
  advantages
• Short life cycle so grows fast prolific
• Normally grows on minimal media, so
  investigators can manipulate fungus growth
  conditions
• Is haploid, so mutations crosses reveal results
  easily
• Can undergo sexual and asexual reproduction.0
• UV irradiation produced mutants that could not
  live unless arginine was added to the nutrient
  medium
• Was an enzyme missing? Beadle and Tatum found
• Many mutants, each missing a single enzyme in the
  arginine pathway
• Each mutant was inherited according to Mendelian
  genetics

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BT Flowchart

• Irradiate (expose to UV radiation) wild-type
  collect spores
• Grow on rich medium with all the nutrients
  necessary to establish a culture
• Shift cultures to minimal medium select for
  mutants incompetent for growth (they did not grow
  on minimal medium)
• Grow on media selectively enriched with nutrients
  (amino acids were supplemented into the media).
• Mutants in pathways that normally produce
  arginine were revealed as cultures that grew only
  with arginine-supplemented media
• Normal Pathway
• Precursor ? Orthithine ? Citrulline ? Arginine

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Arginine Pathway Analysis

• Analysis of metabolic mutants revealed multiple
  enzymes that lead to arginine synthesis in a
  serial manner
• Different stages of loss of function could be
  identified, each associated with only one enzyme
• B T proposed that 1 gene coded for 1 unique
  enzyme
• Thus, the one gene one enzyme hypothesis

56
Linus Pauling, Vernon Ingram Sickle-Cell Anemia

• In sickle-cell anemia, hemoglobin (Hb) has poor
  oxygen affinity
• When Hb does not bind oxygen (usually in veins as
  it leaves an organ), it tends to crystallize,
  which worsens the oxygen-carrying capacity of the
  RBC and causes pain
• Damages RBCs and capillaries
• Patient becomes severely anemic
• Condition is painful and eventually lethal
• Ingram sequenced the hemoglobin N terminus and
  found that there was one change in the protein
  backbone 6 amino acids from the N terminus
• Hba (normal) N - Val His Leu Thr Pro Glu Glu
  Lys
• Hbs (sickle) N - Val His Leu Thr Pro Val
  Glu Lys
• Hb is not an enzyme it is merely a polypeptide
  (protein).
• Thus 1 gene gives rise to 1 polypeptide
  (protein).
• Linus Pauling explained how the RBC changes
• Vernon Ingram Sequenced the gene

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Unit 5A Molecular GeneticsSection 4 Protein
Synthesis
58
RNA Structure

• RNA is made like DNA
• Sugar-phosphate backbone
• -OH at the 2 C on the ribose, vs. deoxyribose in
  DNA
• U substitutes for T
• Can self-associate, just like DNA
• Unlike DNA, RNA is single stranded
• Where it self-associates, U pairs with A G with
  C
• Multiple types
• mRNA Messenger RNA carries information
• tRNA Transfer RNA carries a.a.
• One unique tRNA for each a.a.
• rRNA most prevalent type, in ribosome

59
Central Dogma of Genetics
DNA ? RNA ? Protein
60
Prokaryotic and Eukaryotic Gene Expression

• Prokaryotes lack a nucleus eukaryotes have
  nuclei. So,
• Prokaryotes make RNA and protein in cytoplasm
• Eukaryotes make RNA in the nucleus, protein in
  cytoplasm

61
Transcription and Translation in Prokaryotes and
Eukaryotes

• Eukaryotes
• Transcription, the production of mRNA, occurs in
  the nucleus of eukaryotes
• Translation, the production of protein, occurs in
  the cytoplasm of eukaryotes
• Prokaryotes
• Both transcription and translation occur in the
  cytoplasm of prokaryotes
• This means that prokaryotes can produce protein
  very quickly in response to the need for that
  protein, and provides a mechanism to explain the
  very fast growth of prokaryotic cells

62
Protein Synthesis

• Transcription Conversion of DNA to mRNA
• Occurs in the nucleus
• Transcription Conversion of mRNA to a protein
  with the help of tRNA and rRNA.
• Occurs in the cytoplasm at a ribosome (rRNA)

63
RNA has Complementary Coding Also

• If the template DNA is
• A-T-G-C-T-T-A-A-C-C-G-G-T-T
• The transcribed mRNA is
• U-A-C-G-A-A-U-U-G-G-C-C-A-A

64
But there is only 4 Nucleic Acids and all these
Proteins!!!

• Most biologists are rather poor mathematicians.
  But physicists are skilled at math, and physicist
  George Gamow immediately recognized the problem
  when biologists were stumped.
• He proposed that one just look at a geometric
  progression
• If only one AA per nucleotide 4 AA could be
  coded (41)
• If two base / AA, then 16 (42) a.a. could be
  coded
• If three base / AA, then 64 (43) a.a. could be
  coded
• He concluded that there must be a triplet code
  system the coding unit was to be called a codon
• James Watson, in 1961, caused point mutations to
  show that the concept was completely correct

65
Nirenberg and Matthei, 1961

• Set out to show empirically the basis for the
  triplet codon system
• They synthesized poly-uracil RNA.
  Just a long string of Us
• 3' - U - U - U - U - U - U - U - U - U - U - U
  - U - 5'
• They added this synthetic polyU-RNA to a mixture
  of ribosomes, known to play a role in the
  manufacturing of proteins.
• The resulting polypeptide was all phenylalanines
  a long string of Phes
• Phe-Phe-Phe-Phe-Phe-Phe-Phe-Phe-Phe-Phe
• They concluded that UUU encoded Phe. Colleagues
  continued to work on this problem until 1967,
  when the full codon assignment was worked out

66
AA Codon Chart

• The code indicated here is nearly identical
  in all organisms, prokaryotes and eukaryotes
• Note that there is redundancy in the genetic code
• Often four, and up to six (can you find it?)
  codon sequences encode one amino acid

67
Sunburst AA Coding Circle
68
The Flow of Coding Information

• Central Dogma DNA ? RNA ? Protein
• Proposed by Frances Crick
• The association between DNA, RNA and Protein at
  the molecular level is given in this example
• DNA 3' ACC AAA CCG AGT
• mRNA 5' UGG UUU GGC UCA (complementary to DNA)
• Protein Trp Phe Gly Ser
• The string of amino acids has a direct
  relationship to nucleotide bases in RNA and DNA

69
Practice

• Worksheet

70
Protein Synthesis in BacteriaPart 1 -
Transcription
71
Transcription

• Nucleotide triphosphates are added to the growing
  strand at the 3 end
• Phosphodiester bonds are made by DNA dependent
  RNA polymerases
• Two phosphates are lost from each nucleotide
  triphosphate
• Note the antiparallel, complementary strands

72
Transcription and Translation

• Note that the protein N terminus is made nearest
  the 5 end of the RNA strand.
• mRNA is transcribed in a 5 ? 3 direction
  nucleotides are added to the 3 end also called
  the C terminus

73
Promotors Are Upstream of the Coding Region

• Promoters are DNA sequences that initiate
  transcription and further regulate the
  transcription process
• Promoters are upstream of the transcribed region
  of the DNA thus they are at the 3 end of the
  transcribed region
• The transcribed DNA region is the sense strand
• You only make mRNA off the ahead of the leading
  strand.

74
Steps to Transcription

• DNA Helicase comes in and unwinds the DNA, while
  binding proteins hold the DNA open.
• Topoisomerases unwind the DNA further down the
  line and put it back together to prevent kinking.
  
• RNA Promoter region signals where to begin the
  transcription of mRNA.
• Ribose nucleotides pair with the DNA substituting
  U for T and is controlled by RNA Polymerase III.
• This happens from an upstream (5) region and
  moves downstream (3) region and nucleotides are
  added to the 3 end.
• RNA Poly II checks the bases to make sure they
  are correct.
• At the termination signal (set of DNA bases) RNA
  Polymerase I will break the bonds between the DNA
  and mRNA so that mRNA can leave and go into the
  cytoplasm.

75
Protein Synthesis in BacteriaPart 2 - Translation
76
Translation

• Translation is the production of protein from a
  mRNA template
• Ribosomes make protein
• Pair tRNA and mRNA
• Catalyze a condensation reaction between the ends
  of the amino acids to make the peptide bond
• Protein is released either to the cytoplasm or to
  the endoplasmic reticulum in eukaryotes

77
tRNA

• One tRNA for each amino acid
• Amino acids are attached covalently to the 3 end
  of the tRNA by an enzyme called aminoacyl-TRNA
  synthetases
• The tRNAs carry the amino acids to the ribosome
• Each tRNA has an anticodon of 3 bases that pair
  with the codons of the mRNA
• These are usually polynucleotide chains that are
  about 70-80 nucleotides long.
• Since RNA is single stranded the chain folds on
  itself and creates hydrogen bonds between base
  pairs, therefore creating loops.
• Each tRNA is unique

78
Ribosomes - rRNA

• Do not carry information for protein synthesis.
  They are more of a catalyst for protein
  synthesis.
• Ribosomes come in 2 parts Small, large subunits
• 30S and 50S (eukaryotes)
• 20S and 50S (prokaryotes)
• The ribosome allows for the mRNA to sit down and
  allows for tRNA to come and meet the mRNA and a
  peptide bond formed.
• A Site where the tRNA anticodon comes in to
  meet the mRNA codon.
• P Site This is where the tRNA and mRNA move for
  a peptide bond to be formed between the two.

79
Model of Ribosome tRNA / mRNA / Polypeptide
Complex

• The mRNA passes through a groove between the
  large and small subunits
• The tRNAs enter at the A site.
• The mRNA and tRNA move to the P site and rRNA
  catalyzes the production of the peptide bond

80
Steps in Translation

• 1. Initiation of the translation process
• Starts process of protein production
• 3. Elongation
• The continued addition of amino acids to the
  growing polypeptide chain
• 4. Termination
• The end of translation release of the protein

81
Before Translation Starts
82
The Initiation Complex
83
Elongation Begins

• The initiation codon is AUG which codes for the
  anticodon UAC and brings in the AA
  Methionine.
• Notice that the mRNA is read in a 5 to 3
  direction.

84
The Cyclic Process of Elongation
85
Elongation
86
Termination

• The ribosome hits a STOP codon and the process
  ends.
• A release factor binds the A site, and no more
  tRNAs enter.

87
Overview

• Bases add at the 3' end
• The 5' end correlates to the N terminus of the
  protein
• So, the 3' end is equivalent to the C terminus

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Videos of Protein Synthesis

• http//www.johnkyrk.com/DNAtranscription.html

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Eukaryotic RNA Processing

• mRNA must leave the nucleus and go into the
  cytoplasm
• The parts of the code that actually express
  protein are called exons
• Parts of mRNA segments that are cut out are
  called introns or Junk DNA- introns are
  identified by snRNPs (small nucleotide
  ribonucleicprotein complexes) and taken out.
• Terms apply to DNA as well as RNA
• Bacteria dont have introns
• At the end of the process, RNA is modified
• A 7-methyl G (G-cap) is attached to the 5 end
• RNA has a string of As (poly A) added to the 3
  end
• Both modifications protect RNA from degradation
• Thus, the mature mRNA contains less material than
  the DNA.
• It contains exons and some untranslated 3 and 5
  regions, and the stop codons, but no introns
• mRNA is an expression of the DNA information that
  is relevant to making protein

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Eukaryotic RNA Processing 1

• The 5 end of the new RNA transcript is capped
  with a 7-methylguanosine

91
Eukaryotic RNA Processing 2

• A poly-A tail is added
• Introns are removed

92
Eukaryotic RNA Processing 3

• The transcript is transported out of the nucleus
  to the cytoplasm where it will undergo translation

93
So what is a Gene?

• A modern definition of the gene
• A gene is a nucleotide sequence that carries the
  information needed to produce a specific RNA to
  create a protein.

94
Unit 5A Molecular GeneticsSection 5 Mutations
95
Evolution of the Genetic Coding System

• The genetic code is universal and found virtually
  unchanged in all organisms
• This is remarkable!
• Evolution of the coding mechanisms must have
  occurred very early in evolution
• Prokaryotic and eukaryotic coding is a little
  different because eukaryotic genomes carry much
  nonexpressed DNA

96
Exons and Evolution

• Reason for noncoding regions (exons) is not
  clear.
• However, they could be important to the evolution
  of complex organisms.
• Walter Gilbert (early 1980s) exons code for
  particular protein regions and functions
• Different exons could be mixed to form proteins
  with different domains
• Allows for rapid evolution of proteins
• Evolution by exon shuffling
• Introns might have come about as leftover pieces
  of DNA that arose from transposons (so-called
  jumping genes)
• Introns might be important to the integrity of
  the code, requiring exceptionally precise
  processing to produce good code

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Jumping Genes

• Jumping genes are called transposons.
• They are movable sequences that interfere with
  how DNA is read.
• These transposons turn genes on and off randomly.
• Discovered by Barbara McClintock