Exam 2 Slide 1

1
Protein coat labeled with 35S
DNA labeled with 32P
T2 bacteriophages are labeled with radioactive
isotopes.
The Hershey-Chase Experiment (1952)
Bacteriophages infect bacterial cells.
Conclusion DNA (not protein!) is the genetic
material!
Bacterial cells are agitated to remove protein
coats.
35S radioactivity found in the medium
32P radioactivity foundin the bacterial cells
2
Subunits of DNA/RNA Nucleotides
H
H
H
C
HO
H
C
HO
OH
O
O
OH
H
C
C
H
C
C
H
H
H
C
C
H
C
C
H
H

• A 5-carbon sugar
• Deoxyribose in DNA
• Ribose in RNA.
• A phosphate group
• A nitrogenous base
• Purines
• Adenine, Guanine
• Pyrimidines
• Cytosine, Thymine (DNA) Uracil (RNA)

OH
H
OH
OH
Deoxyribose (DNA only)
Ribose (RNA only)
O
O
P
HO
O
Phosphate
NH2
O
H
C
N
N
C
C
N
C
N
C
H
H
C
C
H
C
C
C
N
N
N
N
H2N
H
H
Guanine
Adenine
Purines
O
NH2
O
H
CH3
H
C
C
C
N
C
H
N
C
H
N
C
C
C
C
C
C
C
H
N
N
N
H
H
O
O
O
H
H
H
Cytosine
Uracil (RNA only)
Thymine (DNA only)
Pyrimidines
3
Erwin Chargaffs Rules

• Amount of A T Amount of G C
• Purines (A and G) Pyrimidines (C and T)

4
Solving the Structure of DNA (1953)
Rosalind Franklin
Franklins X-Ray Diffraction Image
James Watson and Francis Crick
5
DNA Structure

• Two antiparallel chains, bonded and twisted
  together double helix.
• Sugar and phosphate backbone railings
• Nitrogenous bases stairs
• Complementary base-pairing
• A forms two H bonds with T
• C forms three H bonds with G

Hydrogen bonds between nitrogenous bases
O
G
O
C
P
P
O
T
O
A
P
Phospho- diester bond
P
C
O
O
G
P
P
A
T
O
O
P
3' end
5' end
Sugar-phosphate "backbone"
6
The Double Helix
2 nm
5'
3'
T
A
T
A
G
C
C
G
T
A
3.4 nm
G
C
Minor groove
T
A
0.34 nm
G
C
T
A
T
A
Major groove
G
C
C
G
T
A
G
C
3'
5'
7
Numbering the C Atoms in the 5-Carbon Sugar
The Phosphodiester Bond
8
Antiparallel Directionality of DNA Structure
Sugar-phosphate "backbone"
O
P
Hydrogen bonds between nitrogenous bases
O
P
G
C
O
O
C
P
P
G
O
O
P
A
P
T
O
O
P
G
C
P
O
O
OH
T
3' end
A
P
Phosphodiester bond
O
P
5' end
9

• Why Chargaffs Rules? Hydrogen Bonding and
  Complementary Base Pairing
• Results from high electronegativity of O and N
  atoms and partial positive charge on associated H
  atoms

10
Deoxyribose- phosphate backbone
DNA vs. RNA
DNA
P
P
P
P
T
T
T
G
P
G
A

• RNA has ribose instead of deoxyribose.
• RNA uses uracil instead of thymine.
• RNA is usually single-stranded.

P
A
P
C
P
A
P
C
G
A
P
Bases
C
T
P
P
P
P
Hydrogen bonding occurs between base-pairs
P
Ribose- phosphate backbone
RNA
P
P
P
P
C
G
A
P
U
A
P
Bases
U
P
G
11
Central Dogma of Gene Expression
12
Proteins and Protein Structure

• Diverse and critical biochemical roles
• Enzymes catalyze biochemical reactions. (e.g.
  proteases, DNAses)
• Defense detect self vs. other (e.g.
  antibodies)
• Transport move small molecules into and out of
  cells (e.g. ion channels)
• Support structural proteins (e.g. collagen,
  keratin)
• Motion contractile proteins (e.g. actin,
  myosin)
• Regulation control cellular operations (e.g.
  hormones)

13
Amino Acids The Building Blocks of Proteins

• All proteins are polymers of only 20 different
  amino acids.
• Amino acids contain a central carbon (Ca) bound
  to
• an amino group (NH2 NH3 when ionized)
• a carboxyl group (COOH COO when ionized)
• a hydrogen atom (H)
• one of 20 different side chains (R groups) that
  give each amino acid its unique chemical
  properties.

• The 20 common amino acids are grouped into
  classes based on the chemical properties of their
  side chains.

14
A. Hydrophilic Amino Acids with Electrically
Charged Side Chains
15
B. Hydrophilic Amino Acids with Polar but
Uncharged Side Chains
16
C. Hydrophobic Amino Acids with Nonpolar Side
Chains
17
Special Case Amino Acids
18
Linking Amino Acids Together The Peptide Bond

• A protein is composed of one or more long chains
  of amino acids (polypeptides) linked by peptide
  bonds between carboxyl group C of one amino acid
  and the amino group N of the next.

19
The Repetitive Polypeptide Backbone of Proteins
Serine
Tyrosine
Cysteine
Repeating Peptide Backbone
20
Four Levels of Protein Structure

• Primary structure The sequence of amino acids
  along the polypeptide chain.
• Secondary structure Folding caused by hydrogen
  bonding within the polypeptide backbone.
• H-bonds develop between amino and carboxyl groups
  from non-adjacent peptides of the backbone.
• Two types of secondary structure
• Alpha helices
• Beta pleated sheets
• Tertiary structure Folding caused by molecular
  interactions among side chains.
• Quaternary structure Joining of two or more
  polypeptide chains to form a functional protein

21
Primary Structure Amino Acid Sequence

• Amino acid sequence of the enzyme lysozyme
• A single polypeptide chain with 129 amino acids

22
Secondary Structure Hydrogen Bonding Between
Non-Adjacent Peptides of the Backbone
23
Tertiary Structure Molecular Interactions Among
the R-Group (Side Chains)

• Chemical processes responsible for Tertiary
  Structure
• Ionic bonds
• Disulfide bridges (between cysteines)
• Hydrophobic exclusion
• Weak Van der Waals interactions
• Hydrogen bonding

Leucine
Serine
Cysteine Cysteine
Aspartic Acid
Aspartic Acid
Lysine
24
Quaternary Structure Joining of Two or More
Polypeptide Chains
Collagen fibrous protein composed of three
separate polypeptide subunits. Hemoglobin
globular protein composed of four separate
polypeptide chains, two alpha subunits and two
beta subunits, plus non-protein heme groups.
25
The Four Levels of Protein Structure
26
Protein Folding Chaperones and Denaturation

• Protein function highly dependent on proper
  folding and three-dimensional structure.
• Proteins can denature (change shape and lose
  function) when exposed to changes in pH,
  temperature, etc.
• Specialized chaperone proteins help new or
  damaged proteins fold/refold properly.

27
Beadle and Tatum Experiment Genes Specify Enzymes
X rays or ultraviolet light
Wild-type Neurospora
Asexual spores
Minimal medium
Growth on complete medium
Products of one meiosis
Meiosis
Select one of the spores
Grow on complete medium
Test on minimal medium to confirm presence of
mutation
Minimal media supplemented with
Minimal control
Nucleic acid
Choline
Pyridoxine
Riboflavin
Arginine
Niacin
Inositol
Folic acid
p-Amino benzoic acid
Thiamine
28
Beadle and Tatum Experiment One Gene One
Enzyme
29
How Genetic Information is Expressed as Proteins
The Central Dogma of Gene Expression
(messenger RNA)
30
The Triplet Code DNA to RNA to Protein
In RNA, uracil (U) takes place of thymine (T)
31
Characteristics of the Genetic Code

• Linear uses mRNA which is complementary to DNA
  sequence.
• Triplet unit of information is 3
  ribonucleotides.
• Unambiguous each codon specifies only one amino
  acid.
• Degenerate more than one codon exists for most
  amino acids.
• Punctuated there are start and stop codons.
• Commaless there is no punctuation within an
  mRNA sequence its continuous.
• Nonoverlapping any one ribonucleotide is part
  of only one codon.
• Nearly Universal with few exceptions, same code
  used by all organisms.

32
The Genetic Code (in language of mRNA)
33
Gene Expression Transcription and Translation in
Prokaryotes vs. Eukaryotes
Eukaryotes
Prokaryotes
34
Transcription in Prokaryotes

• Transcription initiated when the enzyme RNA
  polymerase binds to promoter binding site on
  coding strand.

• Sigma (s) subunit of RNApol recognizes and binds
  with promoter.
• Begins to unwind DNA at -10 sequence (10 base
  pairs upstream of start site).

35
A Prokaryotic Transcription Bubble
Coding (sense)strand
Template strand
3'
C
C
G
G
A
T
T
A
A
A
A
5'
G
A
G
C
T
T
T
T
T
C
C
A
A
C
G
3'
T
DNA
A
C
T
5'
T
G
A
G
G
C
C
G
A
U
U
C
T
Unwinding
3'
G
C
A
A
T
G
Rewinding
mRNA
RNA polymerase
5'
RNA-DNA hybrid helix
RNApol reads DNA template in 3' to 5' direction
RNApol adds nucleotides to mRNA transcript in 5'
to 3' direction (no primer required, no
proofreading capability). Stop sequences at the
end of the gene cause RNA polymerase to cease
transcription.
Coding strand so-called because it has the
same sequence as the mRNA transcript, except T
takes place of U.
36
Transcription in Eukaryotes
Similar to prokaryotes, but many other proteins
transcription factors are involved.
37
RNA Processing in Eukaryotes The 5 Cap and the
3 Poly-A tail

• Modifications to ends of pre-mRNA transcript
• Protect it from degradation.
• Aid export from the nucleus.
• Facilitate ribosome attachment for translation.

38
Eukaryotic Gene Structure Exons, Introns, and
Post-transcriptional RNA Splicing
39
mRNA Splicing in Eukaryotes snRNPs and the
Spliceosome
Why do Eukaryotes have this odd split-gene
arrangement? Why have introns at all?
snRNPs small Nuclear Ribonucleoproteins
40
Functional Significance of Introns I

• Alternative RNA Splicing a single gene can
  encode more than one kind of polypeptide!
• Example Tissue-specific alternative splicing of
  calcitonin/CGRP mRNA.
• In humans
• 30,000 genes, but
• 120,000 mRNA products!
• About 40 of human genes may show alternative RNA
  splicing.

41
Functional Significance of Introns II
Increases Modularity and Flexibility of the
Eukaryote Genome Because exons often code for
discrete functional domains, new proteins can
evolve by exon shuffling among different genes.
Domains are independently folding regions of a
protein that perform specific functions.
42
Translation The Big Picture
43
The Interpreter for Translation Transfer RNA
(tRNA)

• Two key features of tRNA molecules
• An anticodon that recognizes its complementary
  codon on mRNA.
• An amino acid attachment site that binds the
  amino acid corresponding to the codon it
  recognizes.

44
How Many Different tRNAs Are There?
Given that 61 of the 64 codons specify amino
acids one might predict that there should be
61 different tRNA molecules, each with a unique
anticodon.
In fact, the actual number of tRNAs is smaller
(typically about 45). Because of relaxed
base-pairing rules for the base in the third
position (wobble), some tRNAs can recognize two
or more different codons.
45
tRNA Activating Enzymes Aminoacyl-tRNA
Synthetases

• Twenty different aa-tRNA synthetases, each with
  dual specificity for
• tRNA molecules with particular anticodons (with
  wobble.)
• The corresponding amino acid.
• aa-tRNA synthetases charge tRNAs with their
  proper amino acid.

Trp
Trp
CO
Trp
CO
H2O
OH
O
OH
CO
O
Activating enzyme
ACC
ACC
mRNA
tRNATrp
A
C
C
U
G
G
Anticodon
Aa-tRNA synthetase with dual specificity for
tryptophan and tRNATrp
tRNATrp binds to UGG codon of mRNA
Tryptophan attached to tRNATrp
46
The Machinery of Translation The Ribosome

• Composed of two subunits made up of proteins and
  ribosomal RNA (rRNA).
• Subunits join to form a complete ribosome when
  they attach to a mRNA molecule.

Binding sites for tRNA molecules
47
Translation in Prokaryotes Initiation

• Start codon is AUG, specifying a chemically
  modifed form of methionine (fMet).
• Met-tRNA, small ribosomal subunit, mRNA, and
  initiation factors come together to form the
  initiation complex large ribosomal subunit then
  joins.
• Initiation factors position Met-tRNA in P site of
  ribosome.

48
Translation in Prokaryotes Elongation

• 2nd codon positioned in A site, and the
  corresponding tRNA binds, assisted by elongation
  factors.
• tRNA in P site releases its amino acid large
  subunit catalyzes formation of peptide bond
  between it and the amino acid in the A site.
• Translocation ribosome shifts down the mRNA
  molecule toward 3 end tRNA originally in P site
  is now in E, and tRNA originally in A is now in
  P.Growing polypeptide in P site.
• 1st (empty) tRNA is released from E site.
• Repeat!

Translocation
49
Translation in Prokaryotes Termination

• Elongation of polypeptide continues until a STOP
  codon is encountered at A site.
• STOP codon recognized by a release factor (not a
  tRNA), which causes polypeptide to be released
  from ribosome and tRNA.
• Two ribosomal subunits disassociate.

50
Three Alternative Models of DNA Replication
51
Testing the Three Models The Meselson-Stahl
Experiment (Part I)
Centrifuged in cesium chloride gradient
52
Testing the Three Models The Meselson-Stahl
Experiment (Part II)
First replication
Second replication
53
DNA Replication Cast of Characters

• Helicase unwinds the double helix.
• Primase synthesizes RNA primers to give DNA
  polymerase III a head start.
• Single-strand binding protein stabilizes and
  protects single-stranded DNA.
• DNA gyrase relieves torque as DNA unwinds.
• DNA polymerase III synthesizes DNA in a 5'?3'
  direction.
• DNA polymerase I removes RNA primers and fills
  in gaps.
• DNA ligase joins the ends of DNA fragments.

54
Table 14.2
55
Origin of Replication in Bacteria
Parental DNA duplex
Replication origin
New strand
Template strands
Two daughter DNA duplexes
56
Rules for DNA polymerase III

• Can add nucleotides only in a 5'?3' direction.
• Reads the template in a 3'?5' direction.
• Cannot add nucleotides to naked single-stranded
  DNA requires RNA primers to get started.

57
How Nucleotides are Added 5'?3'
New strand
New strand
Template strand
Template strand
3
3
HO
HO
5
5
C
C
P
P
G
G
O
O
O
O
P
P
T
T
A
A
P
P
O
O
O
O
DNA polymerase III
P
P
T
T
A
A
P
P
O
O
O
O
P
P
C
C
G
G
P
P
O
O
O
O
P
P
OH
A
T
A
3
P
P
P
O
O
O
P
T
P
P
P
P
O
A
A
3
OH
O
O
OH
P
P
Pyropho- sphate
Sugar-phosphate backbone
5
5
58
Replication on the Leading and Lagging Strand
3
DNA polymerase III
Leading strand continuous replication
DNA double helix
5
Okazaki fragment
3
Lagging strand discontinuous replication
Primer
5
59
The DNA Polymerase III Complex

• A dimer two separate polymerase molecules, one
  for each strand.
• a subunit catalyzes DNA polymerization.
• b2 subunit acts as sliding clamp.
• e subunit proofreading and error correction.

b2
b2
e
a
a
e
a subunit
b2 subunit
60
How the Two Subunits of DNA Polymerase III
Replicate Two Strands Simultaneously
3
DNA polymerase III
5
Leading strand
3
RNA primer
5
Lagging strand
5
3
61
DNA Replication Fork in Bacteria The Big Picture
5
3
First subunit of DNA polymerase III
Leading strand
Single-strand binding proteins
Parental DNA helix
Okazaki fragment
RNA primer
3
3
5
5
Helicase
Primase
Lagging strand
Second subunit of DNA polymerase III
DNA ligase
5
DNA polymerase I
3
62
Multiple Origins of Replication in Eukaryotes
Parent strand
Daughter strand
1
Point of separation
2
3
4
63
Summary of Replication

• DNA replication is semiconservative parental
  strands separate, and new complementary strands
  are made for each.
• DNA polymerase III synthesizes DNA adds
  nucleotides 5'?3' (reads template 3'?5'), and
  requires RNA primers to get started.
• Due to its antiparallel nature, DNA synthesis
  proceeds differently on each parental strand.
• Leading strand the new strand is synthesized
  continuously 5'?3' toward the unwinding DNA.
• Lagging strand DNA polymerase III lays down
  100-200 nucleotides (Okazaki fragments), and
  then backs up and repeats as the DNA continues to
  unwind discontinuous replication.

64
Mismatched bases (two purines)
Proofreading by DNA Polymerase III
? subunit of DNA Pol III can remove mismatches3
? 5
65
How Methylation Labels DNA
Methylated DNA loop
Methyl group on template DNA strand
Mismatch

• In bacteria, methyl groups are added to many
  adenosines. However
• this doesnt happen on newly synthesized
  strands until several minutes after DNA has been
  replicated thus, the original template is
  temporarily labeled!

66
Methylation-directed Mismatched Base Repair
1. Where a mismatch occurs, the correct base is
located on the methylated strand the incorrect
base occurs on the unmethylated strand.
Mismatch
2. Enzymes detect mismatch and nick unmethylated
strand.
3. DNA polymerase I excises nucleotides on
unmethylated strand.
4. DNA polymerase I fills in gap in 5'    3'
direction.
5. DNA ligase links new and old nucleotides.
Repaired Mismatch
67
UV-Induced Thymine Dimers Cause DNA to Kink
H
P
O
P
H
N
O
CH2
CH2
O
N
N
O
Thymine
O
N
O
DNA strand with adjacent thymine bases
UV light
H
CH3
Thymine dimer
H
CH3
P
H
P
O
O
H
Kink
N
N
O
CH2
CH2
O
N
O
O
Thymine
N
H
CH3
H
CH3
P
P

• Unless corrected by excision repair, resulting
  kink blocks DNA replication and/or leads to
  replication errors.

68
UV Radiation and Xeroderma Pigmentosum A
Vulnerability of cells to UV light damage
100
Cells from normal individuals (high cell survival)
Percentage of cells surviving
10
Cells from XP patients (low survival)
1
Dose of UV light
69
UV Radiation and Xeroderma Pigmentosum B Ability
of cells to repair UV-induced damage
60
Cells from normal individuals (damaged DNA is
repaired)
50
40
Amount of radioactive thymidine incorporated
(counts per minute)
30
20
Cells from XP patients (DNA repair is defective)
10
0
Dose of UV light
70
DNA Repair Mechanisms Summary

• DNA polymerase III proofreads and corrects
  mismatched nucleotides during replication.
• Excision repair systems scan DNA and correct
  remaining mismatches.
• Repair enzymes in bacteria identify the correct
  template strand by its methyl groups.
• Defects in repair system enzymes are implicated
  in a variety of cancers.

71
Unrepaired Mismatches Can Lead to Mutation
A
A
C
T
G
G
C
Wild type
T
T
G
A
C
C
G
A
A
C
T
G
G
C
A
A
C
T
A
G
C
MUTANT
3'
5'
T
T
G
A
T
C
G
T
T
G
A
T
C
G
A
A
C
T
G
G
C
DNA replication
DNA replication
T
T
G
A
C
C
G
A
A
C
T
G
G
A
A
C
T
G
G
C
C
5'
3'
Wild type
Parental DNA
T
T
G
A
C
C
G
T
T
G
A
C
C
G
First generation progeny
A
A
C
T
G
G
C
Wild type
T
T
G
A
C
C
G
Second generation progeny
72
Types of Point Mutations
3
5 Template
Synonymous (silent) mutation base change does
not result in change in amino acid.
73
Third-Position Changes are Often Silent
Second base
U
C
A
G
UUU
UCU
Phenyl- alanine
UAU
UGU
U
U C A G
Tyrosine
Cysteine
UUC
UCC
UAC
UGC
Serine
UCA
Stop codon
UUA
UAA
UGA
Stop codon
Leucine
UCG
UUG
Tryptophan
UAG
UGG
Stop codon
CGU
CCU
CUU
CAU
U C A G
C
Histidine
CGC
CAC
CCC
CUC
Leucine
Proline
Arginine
First base
CGA
CCA
CUA
CAA
Glutamine
Third base
CUG
CGG
CAG
CCG
AUU
AGU
U C A G
AAU
ACU
Serine
Asparagine
AUC
AGC
Isoleucine
ACC
A
AAC
Theronine
AUA
ACA
AGA
AAA
ACG
Lysine
Arginine
Methionine start codon
AUG
AGG
AAG
GGU
GUU
Aspartic acid
GCU
GAU
U C A G
GGC
G
GUC
GCC
GAC
Valine
Alanine
Glycine
GGA
GUA
GCA
GAA
Glutamic acid
GGG
GUG
GCG
GAG
74
Types of Point Mutations
Nonsense mutation base change results in a stop
codon
3
5 Template
75
Types of Point Mutations
Missense mutation base change results in a
change in amino acid
3
5 Template
76
Nonpolar
(Aromatic)
(Aromatic)
Polar uncharged
(Aromatic)
Charged
77
Transitions and Transversions

• Transitions
• Purine ? Purine (A ? G)
• Pyrimidine ? Pyrimidine (C ? T)
• Transversions
• Purine ? Pyrimidine (A ? T, A ? C)
  (G ? T, G ? C)

78
Abnormal Base Pairing and Transitions
Abnormal Tautomeric Pairings
79
Types of Point Mutations
3
5 Template
Frameshift mutation caused by
insertion/deletion of 1-2 nucleotides
80
Non-Disjunction in Meiosis I or II Leads to
Aneuploidy An Abnormal Number of Chromosomes
81
Alterations of Chromosome Structure
82
Burkitt Lymphoma

• Caused by translocation (swapping) of small
  region of chromosomes 8 and 14. Translocation of
  gene c-myc to chromosome 14 disrupts its normal
  function in regulating cell growth, resulting in
  cancer.

83
Chromosomal Mutations Gene Duplication and
Deletion
Deletion and / or duplication of entire genes are
usually a result of mistakes (e.g., unequal
crossover) during meiosis.
Although gene deletions are almost always
harmful, duplicate genes can acquire other
mutations and take on new functions the
evolution of multi-gene families.
84
Chromosomal Mutations Gene Duplication and the
Evolution of Multigene Families
Duplication and Divergence Is a Major Force in
Evolution
Duplicate genes can diverge and take on modified
functions
85
A Multigene Family The Vertebrate Globin Genes
The globin gene family arose from multiple
duplication and divergence events over the last
500 million years.
86
Another Multigene Family Hox Genes
Hox genes are key developmental regulators that
have duplicated and diverged during animal
evolution.
87
Mutation Summary

• Mutations are permanent, heritable changes in DNA
  sequence or chromosome structure.
• Mutations are caused by either rare spontaneous
  mistakes in DNA replication or environmental
  stresses (radiation, chemical mutagens, etc).
• Although usually harmful, mutations can be silent
  or (rarely) beneficial.
• Mutations are the source of genetic variation
  that allows evolution to occur.

88
How Transcription Factors (Regulatory Proteins)
Bind With DNA via the Major Groove
Major groove
Major groove
H
H
N
N
H
CH3
N
N
H
H
H
H
O
O
N
N
N
N
N
H
H
N
H
H
C
G
A
N
N
N
N
Sugar
N
H
O
O
H
H
Phosphate
Minor groove
Minor groove
Key
Hydrogen bond donors
Hydrogen bond acceptors
Hydrophobic methyl group
Hydrogen atoms unable to form hydrogen bonds
89
Three Major DNA-Binding Motifs of Transcription
Factors
90
Prokaryotic Gene Organization The
Operon(iIlustrated by the lac operon)
Gene for repressor protein
Gene for permease
CAP-binding site
Operator
Promoter for I gene
Gene for b-galactosidase
Promoter for lac operon
Gene for transacetylase
CAP
Plac
O
I
PI
Z
A
Y
Regulatory region
Coding region
lac control system
Operon protein-coding genes and regulatory
sequences that are part of a single expression
unit.
91
Operon Cast of Characters
DNA sequences Promoter DNA sequence to which
RNA polymerase binds. Structural genes encode
the actual proteins produced by the
operon. Operator sequence that overlaps with
the promoter repressor binds to it. Proteins
(transcription factors) Repressor protein that
binds to operator and blocks transcription. Activa
tor protein that enables RNA polymerase to bind
to promoter.
92
Regulatory Control of the trp Operon A
Repressible Operon
Inactive repressor
RNA polymerase
Tryptophan absent
mRNA synthesis
Promoter
Tryptophan is synthesized
Genes are ON
Start of transcription
Operator
Tryptophan present
Tryptophan
Active repressor
RNA polymerase cannot bind
trp operon comprises protein-coding structural
genes and regulatory sequences involved in
synthesis of tryptophan
Tryptophan is not synthesized
Genes are OFF
93
Binding of tryptophan to the trp repressor
produces an allosteric (shape-changing) response,
activating repressor and allowing it to bind with
DNA operator, repressing transcription.
How Tryptophan Activates the trp Repressor
3.4 nm
Tryptophan
Inactive trp repressor protein
Active trp repressor protein
94
The lac Operon
Gene for repressor protein
Gene for permease
CAP-binding site
Operator
Promoter for I gene
Gene for b-galactosidase
Promoter for lac operon
Gene for transacetylase
CAP
Plac
O
I
PI
Z
A
Y
Regulatory region
Coding region
lac control system
95
How the lac Repressor Works I
RNA polymerase
Repressor
Plac
O
Promoter for lac operon
RNA polymerase cannot transcribe lac genes when
repressor bound to operator.
DNA helix
96
How the lac Repressor Works II
RNA polymerase cannot transcribe lac genes when
repressor bound to operator.
RNA polymerase
RNA polymerase
Repressor
Repressor
CAP
A
Plac
Y
O
Plac
Z
O
CAP
Promoter for lac operon
I
Promoter
Operator
cAMP
lac operon is "repressed"
Allolactose (inducer)
RNA polymerase
DNA helix
A
CAP
Y
Plac
Z
O
CAP
I
Promoter
Operator
cAMP
lac operon is "induced"
Allolactose (modified form of lactose) binds with
lac repressor, changing its shape and knocking
repressor off of operator
97
The Activator How CAP Works I
RNA polymerase
CAP-binding site
CAP
Plac
O
Promoter for lac operon

• When glucose low, cAMP binds with CAP, allowing
  it to bind with CAP-binding site on DNA.
• CAP binding is necessary to recruit RNA
  polymerase and initiate transcription

cAMP
CAP
Glucose low, promoter activated
RNA polymerase
CAP
Plac
O
Promoter for lac operon
CAP
Glucose high, promoter not activated
98
The Activator How CAP Works II
CAP
cAMP
99
The lac Operon Putting It All Together
RNA-polymerase binding site (promoter)
CAP- binding site
Operator
Lactose
Glucose
lacZ gene


Operon OFF because CAP is not bound
Repressor


Operon OFF both because lac repressor is bound
and CAP is not
RNA polymerase


CAP
Operon OFF because lac repressor is bound
RNA polymerase


CAP
mRNA synthesis

Operon ON because CAP is bound and lac repressor
is not
100
Structure of Eukaryotic Genes and Transcripts
101
Eukaryotic Transcription Complex
Activators These regulatory proteins bind to DNA
at distant sites known as enhancers. When DNA
folds so that the enhancer is brought into
proximity with the initiation complex, the
activator proteins interact with the complex to
increase the rate
Basal factors These transcription factors
position RNA polymerase at the start of
a protein-coding sequence and then release the
polymerase to transcribe the mRNA
Enhancer
Enhancer
Activator
Enhancer
Activator
Activator
Coactivator
E
F
B
RNA polymerase II
H
TFIID
A
Coding region
T A T A
TATA box
Core promoter and initiation complex
Coactivators These transcription factors transmit
signals from activator proteins to the basal
factors
102
Eukaryotic transcriptional control Cast of
characters DNA sequences
DNA sequences Promoter binds RNA
polymerase TATA box (-25) where 1st
transcription factor binds. Enhancers distant
sites to which activators bind. Silencers also
distant sites (near enhancers) to which
repressors bind. Coding region actual DNA
message to be transcribed
103
Eukaryotic transcriptional control Cast of
characters Proteins
Transcription Factors (Regulatory Proteins) Basal
factors including TATA-binding protein, bind
adjacent to site of transcription
initiation. Coactivators link basal factors
with activators. Activators bind to enhancers
interaction with coactivators basal factors
ensures proper placement of RNA
polymerase. Repressors bind to silencers,
inhibiting transcription. Enzymes RNA Polymerase

104
A Eukaryotic Promoter
Thymidine kinase gene
Thymidine kinase promoter
GC
CAAT
GC
TATA
60 bp
25 bp
80 bp
100 bp
Binding sites for basal transcription factors
Base-pairs
105
The Eukaryotic Initiation Complex
Basal transcription factors
TAFs
RNA polymerase II
E
B
F
TFIID
A
H
T A T A
Coding region
TATA box
Core promoter
106
How Eukaryotic Enhancers Work
107
How Eukaryotic Activators Work
RNA polymerase
Basal transcription factor
Activator
Promoter
Enhancer sequence
Coding region of gene
mRNA synthesis
108
How Eukaryotic Repressors Work

• Repressors may work in several ways
• Compete with an activator on overlapping sites.
• Bind the activation site of an activator,
  preventing its interaction with the transcription
  machinery.
• Directly interact with the transcription
  machinery, blocking assembly.

109
Regulation of Regulatory Proteins

• The activity of a regulatory protein can itself
  be regulated!
• Transcriptionally via control over the quantity
  of protein produced.
• Post-translationally via control over the
  quantity of active (binding-enabled) protein in
  the nucleus.

110
Regulation of Regulatory Proteins
B. Change in conformation by ligand binding. Only
bound protein can bind DNA
F. In order to bind DNA, the protein must first
be translocated to the nucleus
D. Only dimer complex of two proteins can bind
DNA
A. How much protein is created?Transcription,
degradation, translation
C. Change in conformation by phosphorylation.
Only phospho-protein can bind DNA
E. Binding site is revealed only after removal of
an inhibitor
111
Eukaryotic Gene Expression 6 Levels of Control
5. Protein synthesis. Many proteins take part in
the translation process, and regulation of the
availability of any of them alters the rate of
gene expression by speeding or slowing protein
synthesis
4. Destruction of the transcript. Many
enzymes degrade mRNA, and gene expression can
be regulated by modulating the degree to which
the transcript is protected.
DNA
Nuclear membrane
3
3
RNA polymerase
Cap
5

• 1. Initiation of
• transcription.
• Most control of gene
• expression is achieved
• by regulating the
• frequency of
• transcription initiation.

5
Small ribosomal subunit
3
Primary RNA transcript
5
Nuclear pore
5
Large ribosomal subunit
3
Cap
Exons
3
Poly-A tail
mRNA
Poly-A
Introns
tail
mRNA
3. Passage through the nuclear
membrane. Gene expression can be regulated by
controlling access to or efficiency of transport
channels
3
2. RNA splicing. Gene expression can be
controlled by altering the rate of splicing in
eukaryotes.
112
Eukaryotic Gene Expression 6 Levels of Control

• 6. Post-translational
• modification.
• Phosphorylation or
• other chemical
• modifications can
• alter the activity of a
• protein after it is
• produced.

Amino acid
PO4
PO4
Completed polypeptide
tRNA
5
Ribosome moves toward 3 end
Cytoplasm
Ribosome