Lecture 5: Nucleic Acids into Protein' Ch 12 and 13

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Lecture 5 Nucleic Acids into Protein. (Ch 12 and
13)

• Goals
• Introduction to nucleic acids, DNA and
  replication
• Understand how to make a protein (transcription)

Key Terms DNA, RNA, nucleic acid, replication,
polymerase, ligase, transcription, translation,
ribosome, splicing, mRNA, tRNA,
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Introduction to Nucleic Acids
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Mystery of the Hereditary Material

• Originally believed to be an unknown class of
  proteins
• Thinking was
• Heritable traits are diverse
• Molecules encoding traits must be diverse
• Proteins are made of 20 amino acids and are
  structurally diverse

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Structure of the Hereditary Material

• Experiments in the 1950s showed that DNA is the
  hereditary material
• Scientists raced to determine the structure of
  DNA
• 1953 - Watson and Crick proposed that DNA is a
  double helix

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Hershey-Chase Experiment

• 1917, Discovery of Bacteriophage
• Why doesnt everyone die of bacterial dysentary?
• Whats eating the bacteria?
• The MSU story
• 1950, Harold Sadoff U of Ill Micro aerosols
• 1952, Hershey and Chase Experiment

Bacteriophage
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Bacteriophages

• Viruses that infect bacteria
• Consist of protein and DNA
• Inject their hereditary material into bacteria

bacterial cell wall
plasma membrane
cytoplasm
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Hershey Chases Experiments

• Created labeled bacteriophages
• Radioactive sulfur
• Radioactive phosphorus
• Allowed labeled viruses to infect bacteria
• Asked Where are the radioactive labels after
  infection?

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Hershey and Chase Results
virus particle labeled with 35S
virus particle labeled with 32P

• Label protein or DNA with radio isotopes
• Infect bacteria with phage particles
• Sheer off the phage (blender)
• Separate bacteria and phage protein
• Progeny of the phage

bacterial cell (cutaway view)
label outside cell
label inside cell
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Hershey and Chase Results

• Conclusions
• DNA is the infective material not protein
• Strong inference DNA is genetic information

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Radical New View of Life!
Sickle Cell Anemia, A Molecular Disease, Pauling
et al. Science 1949
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Structure of Nucleotides in DNA

• Each nucleotide consists of
• Deoxyribose (5-carbon sugar)
• Phosphate group
• A nitrogen-containing base
• Four bases
• Adenine, Guanine, Thymine, Cytosine

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Nucleotide Bases
ADENINE (A)
GUANINE (G)
phosphate group
deoxyribose
THYMINE (T)
CYTOSINE (C)
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Composition of DNA

• Chargaff showed
• Amount of adenine relative to guanine differs
  among species
• Amount of adenine always equals amount of thymine
  and amount of guanine always equals amount of
  cytosine
• AT and GC

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Rosalind Franklins Work

• Was an expert in x-ray crystallography
• Used this technique to examine DNA fibers
• Concluded that DNA was some sort of helix

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• DNA is a very long molecule and does not
  naturally form long, thin fibres. But it is
  possible to extract DNA from cells in the form of
  a viscous gel if a needle is dipped into the gel
  and slowly wound up, it drags out a DNA fibre in
  which many molecules are lined up parallel to
  each other. The X-ray patterns given by DNA
  fibres show a pair of strong arcs along their
  vertical axis Astbury realised that their
  position indicated a very regular periodicity of
  3.4 along the axis of the fibre and that this
  figure was similar to the thickness of the DNA
  bases he therefore suggested that the bases were
  stacked on top of each other "like a pile of
  pennies". He was quite right, but the well-known
  double helix structure had to await much better
  X-ray pictures (obtained by Wilkins, Franklin and
  colleagues at King's College, London) and the
  realisation by Crick and Watson (in Cambridge)
  that the bases were in pairs, joining two
  backbones running in opposite directions.

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Watson-Crick Model

• DNA consists of two nucleotide strands
• Strands run in opposite directions
• Strands are held together by hydrogen bonds
  between bases
• A binds with T and C with G
• Molecule is a double helix

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Watson-Crick Model
Covalent Bonds
Hydrogen Bonds
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DNA Structure Helps Explain How it Duplicates

• DNA is two nucleotide strands held together by
  hydrogen bonds
• Hydrogen bonds between two strands are easily
  broken
• Each single strand then serves as template for
  new strand

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DNA Replication

• Each parent strand remains intact
• Every DNA molecule is half old and half new

new
new
old
old
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Base Pairing During Replication

• Each old strand serves as the template for
  complementary new strand

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Enzymes in Replication

• Enzymes unwind the two strands
• DNA polymerase attaches complementary nucleotides
  
• DNA ligase fills in gaps
• Enzymes wind two strands together

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A Closer Look at Strand Assembly

• Energy for strand assembly is provided by
  removal of two phosphate groups from free
  nucleotides

newly forming DNA strand
one parent DNA strand
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Continuous and Discontinuous Assembly
Strands can only be assembled in the 5 to 3
direction
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DNA Repair

• Mistakes can occur during replication
• DNA polymerase can read correct sequence from
  complementary strand and, together with DNA
  ligase, can repair mistakes in incorrect strand
• DNA damage from environmental factors

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Cloning

• Making a genetically identical copy of an
  individual
• Is cloning new?
• Natural Clones- Maternal twins
• Synthetic Clones-
• Researchers have been creating clones for decades
• Clones were created by embryo splitting

VIDEO CLIP
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Dolly Cloned from an Adult Cell

• Showed that differentiated cells could be used to
  create clones
• Sheep udder cell was combined with enucleated egg
  cell
• Dolly is genetically identical to the sheep that
  donated the udder cell

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More Clones

• Mice
• Cows
• Pigs
• Goats
• Guar (endangered species)

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Bacteriophages

• Viruses that infect bacteria
• Consist of protein and DNA
• Inject their hereditary material into bacteria

bacterial cell wall
plasma membrane
cytoplasm
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Nucleic Acids Into Proteins
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Steps from DNA to Proteins

• Same two steps produce ALL proteins
• 1) DNA is transcribed to form RNA
• Occurs in the nucleus
• RNA moves into cytoplasm
• 2) RNA is translated to form polypeptide chains,
  which fold to form proteins

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Three Classes of RNAs

• Messenger RNA (mRNA)
• Carries protein-building instruction
• Ribosomal RNA (rRNA)
• Major component of ribosome
• Transfer RNA (tRNA)
• Delivers amino acids to ribosome

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DNA vs. RNA

• DNA Bases

• RNA Bases

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Base Pairing During Transcription

• A new RNA strand can be put together on a DNA
  region according to base-pairing rules
• As in DNA, C pairs with G
• Uracil (U) pairs with adenine (A)

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Transcription DNA Replication

• Like DNA replication
• Nucleotides added in 5 to 3 direction
• Unlike DNA replication
• Only small stretch is template
• RNA polymerase catalyzes nucleotide addition
• Product is a single strand of RNA

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Promoter

• A base sequence in the DNA that signals the start
  of a gene
• For transcription to occur, RNA polymerase must
  first bind to a promoter

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Gene Transcription
DNA to be transcribed unwinds
transcribed DNA winds up again
mRNA transcript
RNA polymerase
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Adding Nucleotides
5
3
growing RNA transcript
5
3
direction of transcription
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Transcript Modification
unit of transcription in a DNA strand
3
5
exon
intron
exon
exon
intron
transcription into pre-mRNA
poly-A tail
cap
5
3
5
3
mature mRNA transcript
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Genetic Code

• Set of 64 base triplets
• Codons
• Nucleotide bases read in blocks of three
• 61 specify amino acids
• 3 stop translation

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Code Is Redundant

• Twenty kinds of amino acids are specified by 61
  codons
• Most amino acids can be specified by more than
  one codon
• Six codons specify leucine
• UUA, UUG, CUU, CUC, CUA, CUG

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Redundant?(Genetic Code Secret Decoder Ring)
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Three Stages of Translation

• Initiation
• Elongation
• Termination

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Key Players in Translation

• Ribosome- Center of action
• The tRNAs
• Start Codon (Met)
• The tRNAs- big cast
• The mRNA- translated script
• Stop codon

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Ribosomes
tunnel
small ribosomal subunit
large ribosomal subunit
intact ribosome
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Binding Sites on Large Subunit
binding site for mRNA
A (second binding site for tRNA)
P (first binding site for tRNA)
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tRNA Structure
codon in mRNA
anticodon in tRNA
tRNA molecules attachment site for amino acid
amino acid
OH
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Initiation

• Initiator tRNA binds to small ribosomal subunit
• Small subunit/tRNA complex attaches to mRNA and
  moves along it to an AUG start codon
• Large ribosomal subunit joins complex

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Elongation

• mRNA passes through ribosomal subunits
• tRNAs deliver amino acids to the ribosomal
  binding site in the order specified by the mRNA
• Peptide bonds form between the amino acids and
  the polypeptide chain grows

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Elongation
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Termination

• A stop codon in the mRNA moves onto the ribosomal
  binding site
• No tRNA has a corresponding anticodon
• Proteins called release factors bind to the
  ribosome
• mRNA and polypeptide are released

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Polysome

• A cluster of many ribosomes translating one mRNA
  transcript
• Transcript threads through the multiple ribosomes
  like the thread of bead necklace
• Allows rapid synthesis of proteins

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What Happens to the New Polypeptides?

• Some just enter the cytoplasm
• Many enter the endoplasmic reticulum and move
  through the cytomembrane system where they are
  modified

Dont Worry About it Till After Test 1 !
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Overview
Transcription
mRNA
rRNA
tRNA
Mature mRNA transcripts
ribosomal subunits
mature tRNA
Translation
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When Things Go Wrong

• Mutations
• Base-Pair Substitutions
• Insertions
• Deletions
• Effect of Mutations on DNA vs. RNA

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Effect of Base-Pair Substitution
original base triplet in a DNA strand
a base substitution within the triplet (red)
As DNA is replicated, proofreading enzymes detect
the mistake and make a substitution for it
POSSIBLE OUTCOMES
OR
One DNA molecule carries the original,
unmutated sequence
The other DNA molecule carries a gene mutation
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Frameshift Mutations

• Insertion
• Extra base added into gene region
• Deletion
• Base removed from gene region
• Both shift the reading frame
• Result in many wrong amino acids

mRNA
PARENTAL DNA
amino acid sequence
ARGININE
GLYCINE
TYROSINE
TRYPTOPHAN
ASPARAGINE
altered mRNA
BASE INSERTION
ARGININE
GLYCINE
LEUCINE
GLUTAMATE
LEUCINE
altered amino acid sequence
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Mutation Rates

• How often do mutations happen
• Cell type
• Gene type
• Only mutations in germ (sex) cells are be passed
  to the next generation
• Mutations in somatic cells stay in the body they
  happen in

Genetic Diseases and Cancers