Announcements

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Announcements

1. Grading of Mendel papers A completeness of
   answer (40) F flow and organization (15) R
   references (10) S spelling and grammar (10).
2. Specifics on reading assignments Ch. 11 Skip,
   p. 304, btm. 309- top 312 Ch. 12 skim 327-328
   skip btm 335-336 skip recombination on 338-341
   Ch. 13

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Review of Last Lecture

• 1. Eukaryotic DNA replication is complex
• 2. The end problem and telomerase aging and
  cancer
• 3. The Genetic Code - theoretical evidence for
  triplet code genetic evidence using mutagens,
  ie. insertions and deletions can cause frameshift
  mutations

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Outline of Lecture 23

1. The Genetic Code - biochemical evidence
2. Transcription

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I. Biochemical Evidence

• 1961 Nirenberg, Matthaei used synthetic mRNAs
  and an in vitro translation system to decipher
  the code.
• Polynucleotide Phosphorylase enzyme links NTPs to
  make RNA without a template
• Homopolymers
• poly(U) codes for Phe-Phe-Phe-Phe-
• poly(A) codes for Lys-Lys-Lys-Lys-
• poly(C) codes for Pro-Pro-Pro-Pro-...

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Repeating Copolymers

• Khorana, early 1960s
• UGUGUGUGUGUGUGUGU...
• Cys-Val-Cys-Val-Cys-Val-...
• Therefore GUG or UGU codes for either Cys or Val
• UUCUUCUUCUUCUUC
• Phe-Phe-Phe-Phe-... or
• Ser-Ser-Ser-Ser- or
• Leu-Leu-Leu-Leu-...

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In Vitro Triplet Binding Assay

• Nirenberg and Leder (1964) mixed all 20 amino
  acids with ribosomes, different RNA triplets
• Ribosomes UAU -gt Tyr binds
• Ribosomes AUA -gt Ile binds
• Ribosomes UUU -gt Phe binds, etc.

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Nucleic Acid to Protein

• How does the information in codons of mRNA get
  translated into amino acids in polypeptides?
• Through adapter molecules tRNA
• tRNA has anticodon that base pairs with the codon
  in mRNA and carries an amino acid corresponding
  to that codon.

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Note that 3rd Base Position is Variable
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Degeneracy and the Wobble Hypothesis

• Codon in mRNA
• Anticodon in tRNA
• Codon 5-1-2-3-3
• Anticodon 3-3-2-1-5
• First two bases of codon are more critical than
  3rd base
• Base-pairing rules are relaxed between 3rd base
  of codon and 1st base of anticodon (third base
  wobble)

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Special Anticodon-Codon Base-Pairing Rules
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II. TranscriptionMaking Sense of the Strands

• DNA coding strand Sense Strand
• DNA template strand Antisense Strand
• mRNA formed Sense Strand

Coding strand
5
3
mRNA 5
3
5
3
Template strand
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Prokaryotic Promoter Lies Just Upstream (5) of
Transcribed Region RNA Polymerase Binds Two
Places
-35 Region
-10 TATA Box
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Initiation of Prokaryotic Transcription Requires
Binding of Sigma Factor to Pol
Note No primer needed
5 to 3
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Termination of Transcription in Prokaryotes

• Occurs when hairpin loops form from
  intramolecular GC base pairing in mRNA.
• Sometimes a special protein called termination
  factor, rho is required for termination.

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Isolating Eukaryotic RNA Polymerases

• Roeder and Futter (1974) Are there enzymes in
  the nucleus that make RNA?
• From cultured frog cells, isolated nuclei.
• Separated proteins by Ion-Exchange Chromatography

Beads with negative charge some proteins bind
strongly, most dont.
Add nuclei, containing proteins
Elute with Na gradient
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Results of Experiment
NaCl Total Protein RNA Synthesis Activity RNA
Synthesis 1 ug/ml ? amanitin
I
II
III
0 10 20 30 40 50
Fraction
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Eukaryotic RNA Polymerases

• RNA Pol I
• 1000 ?g/ml ?-amanitin inhibits
• makes three rRNAs (28S, 18S, 5.8S)
• RNA Pol II
• 1 ?g/ml ?-amanitin inhibits
• makes mRNA and snRNA (small nuclear RNA)
• RNA Pol III
• 50 ?g/ml ?-amanitin inhibits
• makes tRNA and 5S rRNA
• Each recognizes different core promoter regions.

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Anatomy of a Eukaryotic Gene (Protein Encoding)
Pol II, Basal TFs bind
TATA Box
CAAT Box
Cis-regulatory Elements may be located thousands
of bases away Regulatory TFs bind.
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Eukaryotic vs. Prokaryotic Transcription

• In eukaryotes, transcription and translation
  occur in separate compartments.
• In bacteria, mRNA is polycistronic in
  eukaryotes, mRNA is usually monocistronic.
• Polycistronic one mRNA codes for more than one
  polypeptide
• moncistronic one mRNA codes for only one
  polypeptide
• 3 RNA polymerases in euk., 1 in prok. Binding of
  Basal Transcription Factors required for euk. RNA
  Pol II binding.
• Processing of mRNA in eukaryotes
• 5 7-methylguanosine (7mG) cap added
• 3 Poly-A tail added
• Splicing out of introns

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TF
Binding of Eukaryotic RNA Pol II Requires Binding
of Basal TFs to Core Promoter
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RNA Processing in Eukaryotes
Pre-mRNA (primary transcript) 5 cap Poly A
tail Splicing Mature mRNA
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Introns and Exons