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Molecular Biology of the GeneOutline: Molecular Biology of the Gene
• Readings: Chapters 10-12 in Campbell et al• Historical Genetic Material Experiments• Chemical Nature of Nucleic Acids• Structure of DNA• DNA Replication• Gene Expression
– RNA and Protein Synthesis• Gene Technology
Griffith’s Bacterial Transformation Experiments
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Bacteriophage (Phage) Structure
Head
Tail
Tail fiber
DNA
300,
000 ×
Figure 10.1A
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Phage/?Bacterial virus reproductive cycle – Hershey & Chase (1952)
Virus attaches to bacterial cell.
Virus injects DNA.
Virus DNA directs cell to
1. make more phage DNA
2. make protein parts.New phages assemble.
Cell lyses releasing new phages.
Figure 10.1C
Fig. 08.03
Hershey & Chase Phage Reproduction ExperimentsDNA is the Genetic material
2
DNA Structure
1. Nucleotides
2. Nitrogen Bases
3. Key Investigations1. X-Ray Crystallography2. Chargaff’s Rule3. Double Helix of Watson & Crick
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
A
C
T
G
T
Sugar-phosphate backbonePhosphate group
Nitrogenous base
Sugar
A
C
T
G
T
DNA polynucleotide
Phosphategroup
O
O–
OO P C N
N
Nitrogenous base
Sugar(deoxyribose)
DNA Nucleotide
Figure 10.2A DNA is a Nucleotide Polymer
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
C C
CC C
C
O
NC
H
HONH
H3C
H H
H
H
N
N
NH
OC
H HN
H CN
N N
NC
CCC
HH
N
NH
CC N
C HNC N
H C
O
HH
Thymine (T) Cytosine (C) Adenine (A) Guanine (G)
PurinesPyrimidines
Figure 10.2B Four Nitrogenous bases are in DNA
OH
O
PO4
Base
CH2O
Base
OPO
C
O–O
CH2
DNA = Nucleotide PolymerSugar-Phosphate Linkage
Sugar
Phosphate
Sugar
Nucleotide
Nucleotide
Chargaff’s Rule
Base Pair Ratio
A:T G:C1.1:1
1:11:11:11:1
1.1:1
1:11:1
1.1:11:11:11:1
Fig. 14.9ab
X-Ray Crystallography: Rosalind Franklin
DNA was helicalDNA had repetitive elements0.34 nm, 3.4 nm, 2.0nm
3
A
A
T
T
C
C
G
G
Phosphodiesterbond
Sugar-phosphate"backbone"
Watson & CrickDNA Model
Paired Nitrogen
Bases
Double Helix
G C
Major groove
3.4 nm
0.34 nm
2 nm
C GG C
G C
G C
C G
TA
TA
T A
T A
T A
T A
T A
G C
DNA StructureFranklin’s
Crystallography made sense!
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Three representations of DNA
G CT A
A T
GG
CCA T
GC
T AT A
A TA T
G CA T
O
OOH
–O P
O O–O P
O
O OP– O
– O OP
O O
O
OH
H2C
H2C
H2C
H2C
O
O
O
O
O
O
O
O
P O–
O–
O–
O–
OH
HO
O
O
O
P
P
P
O
O
O
O
O
O
O
O
T A
G C
C G
A TCH2
CH2
CH2
CH2
Hydrogen bond
Ribbon model Chemical structure Computer model
Basepair
Figure 10.3D
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Start Monday 11/3/06
DNA discovery (video)
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Semiconservative DNA replication
A TC GG CA TT A
A TC GG CA TT A
A TC GG CA TT A
A TC GG CAT
A T
C G
ACTA
Parental moleculeof DNA
Both parental strands serve as templates
Two identical daughtermolecules of DNA
Nucleotides
1. Unwinding 2. Pairing 3. Joining
Figure 10.4A
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
DNA strands have an opposite orientation
P
P
P
P
P
P
P
P
HO
OH
A
C
G
T
T
C
G
A
2′1′3′
4′
1′ 4′3′
2′
5′ end 3′ end
3′ end 5′ end
Figure 10.5B
5′
5′
DNA replication – A Closer Look
4
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
DNA replication – A Closer LookFigure 10.4B
G C
A T
G C
A TC G
AGA
CG
CG
CG
TAG
C
TAT
AA
TT
A
CG
CG
CG
TA
G
C
T
A
TA
AT
TA
TCT
DNA must unwind
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
3′
5′3′
5′3′
5′
5′3′
Daughter strandsynthesizedcontinuously
Daughter strandsynthesizedin pieces
Parental DNA
DNA ligase
DNA polymerasemolecule
DNA replication – A Closer LookNew DNA strands are synthesized continuously & discontinuously
Figure 10.5C
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
10.5 DNA replication: A closer look• DNA replication
– Begins at specific sites on the double helix
Figure 10.5A
Origin of replication
Two daughter DNA molecules
Parental strand
Daughter strand
Bubble
DNA Synthesis Animation
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Normal Hemoglobin β chain (Sanger 1953)Valine Histidine Leucine Threonine Proline Glutamic
acid
Sickle cell anemia Hemoglobin β chain (Ingram 1956)
Valine Histidine Leucine Threonine Proline Glutamicacid
Glutamicacid
Valine
Genes Specify Sequences of Amino Acids
GENE = Unit of Heredity
GENE = Sequence of nucleotides that determines the amino acid sequence of a protein
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
DNA strand
Transcription
Translation
Polypeptide
RNA
A A A C C G G C A A A A
U U U G G C C G U U U U
Gene 1
Gene 2
Gene 3
DNA molecule
Gene Expression – The Central DogmaFigure 10.7
OneDNA strand
5
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Figure 10.9B Transcription of a gene
RNA polymerase
DNA of genePromoter DNA
1 Initiation
2 Elongation
3 Termination GrowingRNA
Completed RNA
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
RNApolymerase
RNA nucleotides
Direction of transcription
Template Strand of DNANewly made RNA
TC
A T C C A A TT
GG
CC
AATTGGAT
G
U
C A U C C AA
U
Figure 10.9A Transcription of a gene – a closer look
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Gene Expression – The Central Dogma
DNA
Transcription
RNA
Protein
Translation
Figure 10.6A
RNA leaves the nucleus and enters the cytoplasm
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
DNA strand
Transcription
Translation
Polypeptide
RNA
Amino acid
Codon
A A A C C G G C A A A A
U U U G G C C G U U U U
Gene 1
Gene 2
Gene 3
DNA molecule
Gene Expression – DNA is a triplet codeFigure 10.7
OneDNA strand
Codon Codon Codon
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
The genetic code (using RNA codons)
UUUUUC
UAUUACUAA Stop
UAG Stop
UGUUGC
UGA StopUGG Trp
CUUCUCCUACUG
CCUCCCCCACCG
CAUCACCAACAG
CGUCGCCGACGG
GUUGUCGUAGUG
GCUGCCGCAGCG
GAUGACGAAGAG
GGUGGCGGAGGG
ACUACCACAACG
AAUAACAAAAAG
AGUAGCAGAAGG
AUUAUCAUAAUG Met or
START
Phe
Leu
Leu
lle
Val Ala
Thr
Pro
Ser
UCUUCCUCAUCG
Asn
Lys
His
Gln
Asp
Glu
Ser
Arg
Arg
Gly
CysTyr
G
A
C
U
U C A GUCAG
UCAG
UCAG
UCAG
Third base
Second base
First base
UUAUUG
Figure 10.8A
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
An exercise in translating the genetic codeFigure 10.8B
T A C T T C A A A A T C
A T G A A G T T T T A G
A U G A A G U U U U A G
Transcription
Translation
RNA
DNA
Met Lys PhePolypeptide
Startcodon
Stopcodon
Strand to be transcribed
6
Types of RNA
1. Ribosomal RNA rRNA
2. Transfer RNA tRNA
3. Messenger RNA mRNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Anticodon
Anticodon
3’
3’
Transfer RNA Structure & Function
Amino acidattaches here
tRNA red&yellowATP (green)Enzyme (blue)
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Ribosomes build polypeptides• A ribosome consists of two subunits
– Each made up of proteins and a kind of RNA called ribosomal RNA
tRNAmolecules
mRNA Small subunit
Growingpolypeptide
Largesubunit
Figure 10.12A Ribosome Structure
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Subunits of a ribosomeHold tRNA and mRNA close together during translation
Largesubunit
mRNA-binding site
Smallsubunit
tRNA-binding sites
Growing polypeptide
Next amino acid to be added to polypeptide
mRNA
tRNA
CodonsFigure 10.12B, C
Ribosome Structure
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Smallsubunit
Largesubunit
FunctionalRibosome
mRNABinding
site
E P A
Fig. 15.2(TE Art)
Ribosome StructureCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
fMet
E
P site
A 5'
3'
UUA C
A G
Protein Synthesis: Initiation
7
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Elongationfactor
Leu
tRNAfMet
P site
E A
mRNA
5'3'
U UA A AC
CA U UG G
G
AC
Fig. 15.16a(TE Art)
Protein Synthesis: Elongation
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
LeufMet
5'3'
U UA A A
ACC
CA U UG G
G
Fig. 15.16b(TE Art)
Protein Synthesis: Elongation
E
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Leu
fMet
5'3'
U UA A A
ACC
CA U UG G
G
Fig. 15.16c(TE Art)
Protein Synthesis: Translocation
E
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Leu
fMet
5' 3'U UA A AAC
CC
A U UG GG
Fig. 15.16d(TE Art)
Protein Synthesis: Translocation
A
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Leu
Leu Leu LeutRNA
fMet fMetfMet fMet
P site
E site
A site
mRNA
5' 5' 5' 5'3' 3' 3' 3'U UA AAACC
CAU UG GGU UA AAACC
CAU UG GGU UA AAACC
CAU UG GGU UA AACCAU UG G
GAC
Fig. 15.16(TE Art)
Protein Synthesis
Initiation Elongation Translocation
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Val Ser
Ala Trp
Polypeptide chainreleased
tRNA
AA A
C CU UG G5'
3'
Fig. 15.17b(TE Art)
Release Factor
Protein Synthesis: Termination
8
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Val ValSer Ser
AlaAla
TrpTrp
Releasefactor
P siteE
site Asite
mRNA
Polypeptide chainreleased
tRNA Largeribosomalsubunit
Smallribosomalsubunit
ACC
AAACC
U UGGA AACCU UGG5' 5'
3' 3'
tRNA
Fig. 15.17(TE Art)
Protein Synthesis: Termination
Protein Synthesis Animation
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Eukaryotic mRNA is processed before leaving the nucleus
Exon Intron Exon Intron ExonDNA
Cap TranscriptionAddition of cap and tail
RNAtranscript with capand tail Introns removed Tail
Exons spliced togethermRNA
Coding sequenceNucleus
Cytoplasm
mRNA
Figure 10.10
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
• Alternative splicing may generate two or more types of mRNA from the same transcript
DNA
RNAtranscript
mRNA
Exons
or
Figure 11.7Eukaryotic RNA may be spliced in more than one way
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Gene Expression in Prokaryotes
mRNA
Protein
Translation
Transcription
mRNA
IntronDNA
Primary RNA transcript
Protein
Processing5’ 3’
Cap Poly-A tail
Translation
Transcription
Gene Expression in Eukaryotes
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
DNA
mRNA
DNA
Protein
mRNA
Protein
Lactose
Promoter OperatorLactose-utilization genes
repressor
RNA polymerasecannot attach topromoter
RNA polymerasebound to promoter
Inactiverepressor
Enzymes for lactose utilization
Operon turned off(lactose absent)
Operon turned on(lactose inactivates repressor)
Regulatorygene
Figure 11.1B Protein Assemblies Control Gene Expression
9
Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings
Transcription Factors assist in initiating eukaryotic transcriptionEnhancers Promoter
GeneDNA
Activator proteins
Other proteins
Transcriptionfactors
RNA polymerase
Bendingof DNA Transcription
Figure 11.6 Protein Assemblies Control Gene Expression
RNA polymerase
END
Gene Expression