Unit IV DNA, RNA, and Protein Synthesis

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Unit IV- DNA, RNA, and Protein Synthesis
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Importance and Structure of DNA
Deoxyribo-Nucleic Acid

• Historical Review
• 1900s Morgans studies with fruit flies showed
  that genes were located on chromosomes and
  chromosomes consisted of protein and DNA
• 1952- Hershey-Chase demonstrated that DNA (not
  protein) was the genetic material of a viral phage

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Figure 16.2a The Hershey-Chase experiment phages
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Figure 16.2b The Hershey-Chase experiment
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Phages Infecting a bacterium
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Figure 16.1 Transformation of bacteria
Griffith (and later Avery, McCarty and MacLeod)
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The Structure of DNA

• Nucleotide monomers
• Phosphate
• Pentose Sugar (C5) Deoxyribose Sugar
• Organic Nitrogen Base
• Cytosine (C)
• Adenine (A)
• Guanine (G)
• Thymine (T)

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Structure of DNA cont

• Polynucleotide chain with linkage via phosphates
  to next sugar, with nitrogen base away from
  backbone of
• Phos-sugar-phos-sugar
• Dehydration synthesis

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Beginning of the 1950s several labs were
studying the structure of DNA

• Maurice Wilkins Rosalind Franklin
• X-ray crystallography x-rays pass through pure
  DNA and defraction of x-rays were then examined
  on film
• James Watson and Francis Crick did not have the
  expertise of Franklin and were without proper
  photos until

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Figure 16.4 Rosalind Franklin and her X-ray
diffraction photo of DNA
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Watson and Crick
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April 1953 Classical one page paper in Nature
by Watson and Crick

• A double helix 2 polynucleotide strands
• Sugar-phosphate chains of each strand are like
  the side ropes of a rope ladder
• Paris of nitrogen bases, one from each strand,
  form the rungs or steps
• The ladder forms a twist every 10 bases (all from
  x-ray studies!)

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Figure 16.5 The double helix
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Internal Structure of DNA Purine and
pyridimine? REMEMBER X-RAY DATA
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Confirms Erwin Chargaffs Rules

• of Adenine to of thymine
• of guanine equal to of cytosine
• This dictates the combinations of N-bases that
  form steps/rungs
• Does not restrict the sequence of bases along
  each DNA strand

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Replication/Duplication of DNA

• Due to complimentary base paring one strand of
  DNA determines the sequence of the other strand
• Therefore, each strand of double stranded DNA
  acts as a template
• The double helix first unwinds controlled by
  enzymes and uses new nucleotides that are free
  in the nucleus to copy a complimentary strand off
  the original DNA strand

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Figure 16.7 A model for DNA replication the
basic concept (Layer 1)
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Figure 16.7 A model for DNA replication the
basic concept (Layer 2)
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Figure 16.7 A model for DNA replication the
basic concept (Layer 3)
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Figure 16.7 A model for DNA replication the
basic concept (Layer 4)
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Information storage in DNA

• The 4 nitrogenous bases are the alphabet or
  code for all the traits the organism possesses
• Different genes or traits vary the sequence and
  length of the bases
• ATTTCGGAC vs. GGGATTCTAG vs. GATC

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There are a series of enzymes that control DNA
replication enzymes which

• Uncoil the original double helix strand via a
  helicase
• Single-strand binding protein keeps helix apart
  so replication can start
• Prime an area to start replication primase
  except it adds RNA nucleotides at first
• Polymerase to join individual nucleotides
  (dehydration synthesis)
• Ligases to join short segments

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Figure 16.8 Three alternative models of DNA
replication
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DNA REPLICATION
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Figure 16.9 The Meselson-Stahl experiment tested
three models of DNA replication (Layer 1)
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Figure 16.9 The Meselson-Stahl experiment tested
three models of DNA replication (Layer 2)
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Figure 16.9 The Meselson-Stahl experiment tested
three models of DNA replication (Layer 3)
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Figure 16.9 The Meselson-Stahl experiment tested
three models of DNA replication (Layer 4)
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Figure 16.10 Origins of replication in eukaryotes
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Figure 16.11 Incorporation of a nucleotide into
a DNA strand
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Antiparallel Arrangement of Double Strands

• The carbons of the deoxyribose sugar are numbered
• 3 carbon attached to an -OH group
• 5 carbon holds the phosphate molecule of that
  nucleotide
• 3 ready to bond with another nucleotide to form
  a polynucleotide link (5 to3)
• Notice complimentary strand in opposite direction
  (5 to 3)
• DNA always grows 5 to 3 never 3 to 5

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Definitions

• Origins of Replication where replication of the
  DNA molecule begins
• Bacteria circular DNA 1 origin of replication
  (RF)
• Eukaryotes multiple origins of replication
  (ORFS)
• ORF Replication Fork

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More Definitions

• DNA Polymerases enzymes that catalyze DNA
  replication
• Leading Strand Synthesized continuously towards
  the replication fork by the DNA polymerase in one
  long fashion
• Lagging Strand Synthesized by short fragments
  away from the replication fork by the DNA
  polymerase

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Definitions Cont

• Ligase combines (joins) short fragments
• Primer starts replication of DNA (in this case
  its RNA)
• Primase an enzyme that joins the RNA
  nucleotides to make the primer
• Helicase an enzyme that untwists the double
  helix at the replication fork
• Nuclease a DNA cutting enzyme

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DNA REPLICATION -VIDEO
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Figure 16.13 Synthesis of leading and lagging
strands during DNA replication
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Figure 16.14 Priming DNA synthesis with RNA
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Figure 16.15 The main proteins of DNA
replication and their functions
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Figure 16.16 A summary of DNA replication
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Figure 16.17 Nucleotide excision repair of DNA
damage
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A PROBLEM!

• The end of the leading strand was initiated with
  an RNA primer
• Normally removed by other DNA polymerase
• Removal of gaps by DNA Polymerase doesnt work on
  lagging strand end
• RNA primer removed with no replacement
• A GAP!
• SHORTER AND SHORTER FRAGMENTS?

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Prokaryotes have circular DNA no problem at
ends (there arent ANY!

• Eukaryotes have special terminal sequences of 6
  nucleotides that repeat from 100-1000 times with
  no genes included
• Telomers
• Protect more internal gene materials from being
  eroded
• Germ cells / sex cells have a special enzyme
  (telomerase) that actually restore shortened
  telomers
• Somatic cells telomer continues to shorten and
  may play a role in aged cell death
• Cancer cells
• A telomerase prevents very short lengths

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Figure 16.19a Telomeres and telomerase
Telomeres of mouse chromosomes
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Ribonucleic Acid (RNA)

• Structure of RNA
• Nucleotide monomer
• Phosphate
• Pentose sugar ribose (extra oxygen)
• Nitrogenous base (A/G/C/U)
• Single stranded
• 3 types (mRNA, tRNA, rRNA)

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Synthesis of RNA - transcription

• DNA acts as a template, but only one strand of
  DNA utilized at a given time
• This exposed strand is controlled by specific
  enzymes that pair the DNA nucleotides with free
  RNA nucleotides which are also present in the
  nucleus
• These RNA nucleotides form a single stranded RNA
  nucleic acid
• DNA ATTGGCT
• RNA UAACCGA
• Short segments of DNA are transcribed at a time
  with start and stop messages

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Figure 17.2 Overview the roles of transcription
and translation in the flow of genetic
information (Layer 1)
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Figure 17.2 Overview the roles of transcription
and translation in the flow of genetic
information (Layer 2)
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Figure 17.2 Overview the roles of transcription
and translation in the flow of genetic
information (Layer 3)
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Figure 17.2 Overview the roles of transcription
and translation in the flow of genetic
information (Layer 4)
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Figure 17.2 Overview the roles of transcription
and translation in the flow of genetic
information (Layer 5)
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Figure 17.6 The stages of transcription
initiation, elongation, and termination (Layer 1)
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Figure 17.6 The stages of transcription
initiation, elongation, and termination (Layer 2)
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Figure 17.6 The stages of transcription
initiation, elongation, and termination (Layer 3)
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Figure 17.6 The stages of transcription
initiation, elongation, and termination (Layer 4)
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Figure 17.6 The stages of transcription
elongation
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Three types of RNA

• mRNA messenger RNA
• Transcribed from a specific segment of DNA which
  represents a specific gene or genetic unit
• tRNA transfer RNA
• Transcribed from different segments of DNA and
  their function is to find a specific amino acid
  in the cytoplasm and bring it to the mRNA
• rRNA ribosomal RNA
• Transcribed at the nucleolus - with proteins
  function as the site of protein synthesis

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Three types of RNA
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Protein Synthesis Translation

• Ribosomes sites of protein synthesis
• 30 - 40 protein
• 60 - 70 RNA (rRNA)
• Assembled in nucleus and exported via nuclear
  pores
• Antibiotics can paralyze bacterial ribosomes, but
  not eukaryotic ribosomes (not targeting them)
• 2 ribosomal subunits a large and a small
• Small subunit has been used as a means of
  classifying different bacteria and different
  invertebrates (16S)
• Eukaryotes 18S
• There are three sites on the ribosome that are
  involved in protein synthesis

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Ribosomes bring mRNA together with amino acid
bearing tRNAs

• Three ribosomal sites
• P Site (peptidyl-tRNA) holds the tRNA carrying
  the growing peptide chain after several amino
  acids have been added
• A site (aminoaccyl-tRNA) holds the next single
  amino acid to be added to the chain
• E site (exit site) site where discharged tRNA
  minus amino acids leave ribosome

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Figure 17.15 Translation the basic concept
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Preparation of Eukaryotic mRNA

• RNA splicing- a cut and paste job to remove
  nucleotides from transcribed mRNA
• 8000 nucleotides transcribed but the average gene
  contains 1200 nucleotides
• Long non-coding segments (introns) interspersed
  between coding segments (exons) expressed via
  amino acids

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(No Transcript)
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Figure 17.17 The initiation of translation
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Figure 17.18 The elongation cycle of translation
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Protein Synthesis (cont)Initiation elongation
- termination

• Starting at one end of the mRNA, the small
  ribosomal subunit associates with the mRNA and
  accepts the first tRNA with its activated amino
  acid attached Initiation
• tRNA associate with a triplet codon exposed on
  the mRNA these are 3 nitrogenous bases that
  bond with 3 complementary bases exposed
  (anticodon) on the tRNA opposite the attached
  amino acid
• Wobble
• Arent 61 tRNAs, are 54tRNAs

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Figure 17.4 The dictionary of the genetic code
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Figure 17.3 The triplet code
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tRNA complexes with its amino acid in the
cytoplasm using ATP activated tRNA

• The activated tRNA-amino acid complex moves
  towards the ribosomal area and find a triplet
  codon exposed that is complementary to the
  anticodon of the tRNA
• The first activated tRNA-amino acid, after its
  anticodon is bound to the mRNA codon, associates
  with the large ribosomal subunit which now joins
  the smaller subunit and the mRNA and the tRNA
  (TAKE A BREATH!)

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The first tRNA and its amino acid now occupy the
P site of the large ribosomal subunit

• Review at this point the 2 part ribosome is
  assembled, the mRNA has started to be read, and
  one tRNA plus amino acid is occupying the P site
• That means the adjacent A site is free to accept
  a second activated tRNA and its amino acid, but
  only if the anticodon of this tRNA matches the
  next three base pairs exposed (codon)

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Protein Synthesis continued

• At this point, there are 2 tRNA-amino acid
  complexes adjacent to each other Elongation
  involved one amino acid being added in a three
  step process
• Codon recognition the mRNA codon in the A site
  matches with the anticodon of the tRNA amino
  acid complex
• Peptide bond formation between the new amino acid
  in the A site and the amino acid (later peptide)
  in the P site
• Translocation

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Translocation the ribosome moves the tRNA into
the A site, and its attached peptide to the P
site, as the previous tRNA from the P site moves
to the E (Exit) site and leaves the ribosome

• Review once this process is under way, an
  activated tRNA with its amino acid finds an
  exposed codon in the A site, attaches via
  H-bonds, then forms a peptide bond with the
  polypeptide associated with the tRNA sitting in
  the adjacent P site. For a moment, the longer
  polypeptide chain is only attached to the tRNA in
  the A site. Now the entire ribosome shifts so
  that the

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Yet More Protein Synthesis

• The empty tRNA from the P site moves in to the E
  site and leaves the ribosome
• As the tRNA with the polypeptide chain moves from
  the A site to the now empty P site .exposing a
  new codon.
• GUESS WHAT HAPPENS NEXT?!

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A question?

• Every time a new codon is exposed in the A site,
  a specific tRNA-AA complex moves into the site.
  What originally terermined this mRNA Codon

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The Answer!

• The original DNA that was transcribed
• This elongation of 1 AA takes about 0.1 s
• Termination the above continues (dozens to
  hundreds or more AA added) until the STOP CODON
  is reached (codon at the end of the mRNA)
• This codon does not have a matching tRNA
  anticodon so the tRNA-AA attaches in the A site
  and the tRNA moves to the E site and releases the
  polypeptide chain

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FIANALLY - SUMMARY

• The take home message
• At the ribosome, the genetic language of DNA is
  translated into a different language Via RNA
  into the functioning language of PROTEINS!!!!

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Figure 17.17 The initiation of translation
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Figure 17.18 The elongation cycle of translation
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Figure 17.19 The termination of translation
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Figure 17.20 Polyribosomes
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Table 17.1 Types of RNA in a Eukaryotic Cell
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Figure 17.23 The molecular basis of sickle-cell
disease a point mutation
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Figure 17.24 Categories and consequences of
point mutations Base-pair insertion or deletion
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Figure 17.24 Categories and consequences of
point mutations Base-pair substitution
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Figure 17.25 A summary of transcription and
translation in a eukaryotic cell
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Figure 18.19 Regulation of a metabolic pathway
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Control of Protein SynthesisRegulation of Gene
Expression

• Every cell has the same numbers and types of
  chromosomes
• Development and normal gene function requires
  precise gene expression in an on and off manner
• Operon cluster of gene segments on DNA and its
  controlling segments
• Repressible
• Inducible

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Regions of the Operon (DNA)

• Promoter region promotes transcription by
  binding with RNA polymerase
• Operator region binds a regulatory protein or
  chemical
• Overlaps with the RNA polymerase binding site
• Structural genes code for a particular peptide
  or several peptides
• Start or stop codes

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Figure 18.20a The trp operon REPRESSIBLE
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Figure 18.21a The lac operon INDUCIBLE
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Figure 19.7 Opportunities for the control of
gene expression in eukaryotic cells