25.1 dna structure and replication alfred hershey and martha chase (1952) –demonstrated that dna...
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25.1 DNA Structure and Replication
• Alfred Hershey and Martha Chase (1952)– Demonstrated that DNA (deoxyribonucleic acid) is the
genetic material• Not proteins
– Two experiments• Viral DNA labeled with 32P found in the bacteria and not the
medium – it had entered the bacteria• Viral protein in capsids labeled with 35S found in medium and
not in the bacterium – it never entered the bacteria• Only the DNA entered the bacteria
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centrifuge
virus
1. When bacteria and viruses are cultured together, radioacti veviral DNA enters bacteria.
2. Agitation in blender dislodges viruses. Radioact vity stays inside bacteria.
Viruses inliquid are notradioactive.Bacteria insediment areradioactive.
3. Centrifugation separates viruses from bacteria and allows investigator to detect location of radioactivity.
capsid
centrifuge
DNAlabeledWith 32P
bacterium
capsidlabeledWith 32S
1. When bacteria and viruses are cultured together, radioactive viral capsids stay outside bacteria.
b. Viral capsid is labeled (yellow).
2. Agitation in blender dislodges viruses. Radioactivity stays outside bacteria.
3. Centrifugation separates viruses from bacteria and allows investigator to detect location of rdioactivity.
V iruses inliquid areradioactive.Bacteria insedimentare notradioactive.
a. Viral DNA is labeled (yellow).
Hershey-Chase Experiments
25.1 DNA Structure and Replication
• Structure of DNA– James Watson and Francis Crick determined
the structure of DNA in 1953– DNA is a chain of nucleotides– Each nucleotide is a complex of three
subunits• Phosphoric acid (phosphate)• A pentose sugar (deoxyribose)• A nitrogen-containing base
25.1 DNA Structure and Replication
• Structure of DNA– Four Possible Bases
• Adenine (A) - a purine• Guanine (G) - a purine• Thymine (T) - a pyrimidine• Cytosine (C) - a pyrimidine
– Complimentary Base Pairing• Adenine (A) always pairs with Thymine (T)• Guanine (G) always pairs with Cytosine (C)
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P
SS
S
S
P
S
P
S
P
P
S
P
S
P
S
S
S
S
a. Double helix c. One pair of bases
P
S
P
P
P
P
P
OC
CC
purine basepyrimidine basephosphate3' end5' end
3' end5' end
5'1'
3' 2'
5'
4' 1'
1'
3' 2'
4'
2' 3'
5'
4'
deoxyribose
b. Ladder structure
3' end 5' end
C C
Overview of DNA Structure
25.1 DNA Structure and Replication
• Replication of DNA– Semi-conservative replication
• Each daughter DNA molecule consists of one new chain of nucleotides and one from the parent DNA molecule
– The two daughter DNA molecules will be identical to the parent molecule
25.1 DNA Structure and Replication
• Replication of DNA1. Before replication begins, the two strands of the parent
molecule are hydrogen-bonded together
2. Enzyme DNA helicase unwinds and “unzips” the double-stranded DNA
3. New complementary DNA nucleotides fit into place along separated strands by complementary base pairing. These are positioned and joined by DNA polymerase.
4. DNA ligase seals any breaks in the sugar-phosphate backbone
5. The two double helix molecules identical to each other and to the original DNA molecule
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daughter DNA double helix
daughter DNA double helix
A
A
A
A
A
AA
A
AA
A
A
A
A
A
A
T
T
T
TT
T
T
T
TT
T
G
G
G
G
G
G
G
G
GG
G
G
C
CC
C
C
C
C
CC
C
5' 3'
G
A
5'
3'
region of parentalDNA double helix
region ofreplication:New nucleotidesare pairingwith those ofparental strands
region ofcompletedreplication
oldstrand
newstrand
newstrand
oldstrand
Overview of DNA Replication
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Parental DNA molecule containsso-called old strandshydrogen-bonded by complementarybase pairing.
Region of replication. Parental DNAis unwound and unzipped. Newnucleotides are pairing with thosein old strands.
Replication is complete. Eachdouble helix is composed of an old(parental) strand and a new(daughter) strand.
Ladder Configuration and DNA Replication
25.2 RNA Structure and Function
• RNA (ribonucleic acid)– Uracil (U) used instead of thymine (T)
– Helper to DNA
– Three major types
G
U
A
C
S
S
S
S
P
P
P
P
base isuracil insteadof thymine
one nucleotideribose
Structure of RNACopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Comparison of DNA and RNA
25.2 RNA Structure and Function
• Three Classes of RNA– Messenger RNA (mRNA)
• Produced in nucleus during transcription• Takes a message from DNA to the ribosomes
– Transfer RNA (tRNA)• Produced in nucleus• Transfers amino acids to ribosomes• Each tRNA carries only one type of amino acid
– Ribosomal RNA (rRNA)• Produced in nucleus• Makes up ribosomes (along with proteins)
25.3 Gene Expression
• Gene Expression Requires Two Steps1. Transcription
• Takes place in the nucleus• Portion of DNA serves as a template for mRNA formation
2. Translation• Takes place in the cytoplasm• Sequence of mRNA bases (which are complementary to
those in the template DNA) determines the sequence of amino acids in a polypeptide
25.3 Gene Expression
• Transcription– During transcription, a gene (segment of the DNA)
serves as a template for the production of an RNA molecule
• All three classes of RNA
– Messenger RNA (mRNA)• RNA polymerase binds to a promoter (in DNA)• DNA helix is opened so complementary base pairing can
occur• RNA polymerase joins new RNA nucleotides in a sequence
complementary to that on the DNA
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RNApolymerase
DNATemplatestrand
mRNAtranscript
To RNA processing
3'
5'
Transcription of DNA to Form mRNA
25.3 Gene Expression
• Transcription– Processing of mRNA
• Primary mRNA contains bases complementary to both intron and exon segments of DNA
– Introns are intragene segments - removed– Exons are the portion of a gene that is expressed
• Intron sequences are removed• Guanine cap and poly-A tail added• Mature mRNA transcript ready
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e ee i i
e ee i i
DNA
e ee
(cut-out) (cut-out)
DNA to be transcribed
transcription
poly-Atail
e = exonsi = introns
maturemRNA
primaryRNA
cap
mRNA Processing
25.3 Gene Expression
• Translation– Gene expression leads to protein synthesis– The Genetic Code
• Triplet code - each three-nucleotide unit of a mRNA molecule is called a codon
• There are 64 different mRNA codons– 61 code for particular amino acids
• Redundant code - some amino acids have numerous code words
• Provides some protection against mutations– three are stop codons that signal polypeptide termination
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Second Base
U C A G
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
ThirdBase
FirstBase
UUUphenylalanine
UUCphenylalanine
UUAleucine
UUGleucine
CUUleucine
CUCleucine
CUAleucine
CUGleucine
AUUisoleucine
AUCisoleucine
AUAisoleucine
AUG (start)methionine
GUUvaline
GUCvaline
GUAvaline
GUGvaline
GCGalanine
GCAalanine
GCCalanine
GCUalanine
ACGthreonine
ACAthreonine
ACCthreonine
ACUthreonine
CCGproline
CCAproline
CCCproline
CCUproline
UCGserine
UCAserine
UCCserine
UCUserine
UAUtyrosine
UGUcysteine
UGCcysteine
UACtyrosine
UGAstop
UAAstop
UGGtryptophan
UAGstop
CGUarginine
CAUhistidine
CGCarginine
CAChistidine
CGAarginine
CAAglutamine
CGGarginine
CAGglutamine
AGUserine
AAUasparagine
AGCserine
AGAarginine
AGGarginine
GGUglycine
GGCglycine
GGAglycine
GGGglycine
GAGglutamate
GAAglutamate
GACaspartate
GAUaspartate
AAGlysine
AAAlysine
AACasparagine
Messenger RNA Codons
• Transfer RNA– tRNA transports amino acids to the ribosomes– Boot-like shape– Amino acid binds to one end, the opposite end has an
anticodon• Triplet of three bases complementary to a specific
codon of mRNA– Order of mRNA codons determines the order in which
tRNA brings in amino acids
25.3 Gene Expression
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arginine
G
C
U
anticodon
anticodon
amino acid
a.
amino acid
b.
Transfer RNA: Amino Acid Carrier
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G
G
CC
G
G
C
C
G
G
C G
C
G
C
G
C
C
G
A
U
T
A
A
T
T
A
U
T
G
C
G
DNA
mRNA
codon 1 codon 2 codon 3
anticodon 1GCC
anticodon 2 anticodon 3
polypeptide N N NC C C C C C
arginine
O O O
tryptophantyrosine
trnslationat ribosome
transcriptioninnucleus
DNAdouble helix
ACCAUG
R1 R2 R3
Overview of Gene Expression
25.3 Gene Expression
• Ribosome and Ribosomal RNA– Free or attached to endoplasmic reticulum– Two subunits – one large and one small– Binding site for mRNA and three tRNAs– Binding sites facilitate complementary base pairing
between tRNA anticodons and mRNA codons– Brings amino acids in line in a specific order to form a
polypeptide– Several ribosomes may move along the same mRNA
• Multiple copies of a polypeptide may be made• The entire complex is called a polyribosome
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mRNA
polypeptide
mRNA
large subunit
small subunit
a. Structure of aribosome
tRNA bindingsites
out goingtRNA
b . Binding sites of ribosome
incomingtRNA
c. Function of ribosomes d. Polyribosome
5' 3'
D: Courtesy Alexander Rich;
Polyribosome Structure and Function
25.3 Gene Expression
• Translation Requires Three Steps– Initiation (requires energy)
• Initiation factors assemble components• Start codon (AUG)• P (peptide) site, A (amino acid) site, and E (exit)
site– Elongation (requires energy)– Termination
• Stop codon• Requires release factor
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UC
A
U G G
3'5'
met
asp
ala
ser
3'5'
met
asp
ala
ser thr
3'5'
anticodon
tRNAmet
asp
ala
ser
3'5'
met
ala
ser
AUG
C U G
G A C AUG G A C
4. The ribosome moves for ward; the “empty” tRNA exits from the E site; the next amino acid–tRNA complex is approaching the ribosome.
3. Peptide bond formation attaches the peptide chain to the newly arrived amino acid.
2. Two tRNAs can be ta a ribosome at one time; the anticodons are paired to the codons.
Elongation
1. A tRNA–amino acid approaches the ribosome and binds at the A site.
peptidebond
tryp
val val
tryp
tryp
val
tryp
val
AUG G A C G A CAUG A C CC U GUC A C U GUC A UC A C U G
asp
Elongation
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stop codon
UU
AG A
U G A
UC
UThe ribosome comes to a stopcodon on the mRNA. A releasefactor binds to the site.
release factor
The release factor hydrolyzes the bondbetween the last tRNA at the P site andthe polypeptide, releasing them. Theribosomal subunits dissociate.
Termination
3'
3'
5'
5'
asp
ala
tryp
val
glu
glu
ala
asp
tryp
val
AA U G A
Termination
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DNA
mRNA
anticodon
tRNA
peptide
codonribosome
5'
C CG G
GCG
CG
C
CCC
GUA
CUA
UA
UA
UUA A
TRANSCRIPTION
3. mRNA moves into cytoplasm and becomes associated with ribosomes.
4. tRNAs with anticodons carry amino acids to mRNA.
5. Anticodon–codon complementary base pairing occurs.
6. Polypeptide synthesis takes place one amino acid at a time.
1. DNA in nucleus serves as a template for mRNA.
2. mRNA is processed before leaving the nucleus.
aminoacids
primarymRNA
int rons
nuclearpoer
TRANSLATION
3'5'
3'maturemRNA
large and smallribosomal subunits
Review of Gene Expression
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Housekeeping
Hemoglobin
Insulin
Myosin
Gene type
Cell type Red blood Muscle Pancreatic
25.4 Control of Gene Expression• Housekeeping genes govern functions that are common
to many types of cells– Active in many cell types
• Only certain genes are active in cells that perform specialized functions
• Gene expression is controlled in a cell and this control accounts for its specialization
25.4 Control of Gene Expression
• Gene Expression in Prokaryotes– Operon: Cluster of genes usually coding for proteins
related to a particular metabolic pathway, along with the short DNA sequences that coordinately control their transcription
• Sequences consists of:– Promoter: A sequence of DNA where
transcription begins– Operator: A sequence of DNA where a
repressor protein binds
25.4 Control of Gene Expression
• Gene Expression in Bacteria
– Example: The lac Operon in E. coli
• When lactose is absent:– The regulator gene codes for a repressor that is normally
active
– A repressor protein binds to the operator
– RNA polymerase cannot transcribe the three structural
genes of the operon (the structural genes are not
expressed)
25.4 Control of Gene Expression
• Gene Expression in Bacteria– Example: The lac Operon in E. coli
• When lactose is present:– Lactose binds with the lac repressor– Repressor is unable to bind to the operator– Structural genes are transcribed
• Enzymes are produced
• lac operon is considered an inducible operon– Only activated when lactose induces its expression
• Repressible operons are usually active until a repressor turns them off
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DNA
mRNA
lactose
DNA
RNA polymerase cannot bind to promoter.
regulator gene promoter operator structural genes
Active repressor
3'
act ive repressor
a. Lactose absent. Enzymes needed to metaboliz leactose are not produced.
5'
5'
3'5'
3' RNA polymerase can bind to promoter.
active repressor
b . Lactose present. Enzymes needed to met a bolize lactose are produced only when lactose is present.
enzymes forlactose metabolism
inact ive repressor
The lac Operon
25.4 Control of Gene Expression
• In bacteria:– Single promoter serves several genes that make up
a transcription unit• In eukaryotes, each gene has its own promoter• Bacteria rely mostly on transcriptional control• Eukaryotes employ a variety of mechanisms to
regulate gene expression– Affect whether the gene is expressed, the speed with
which it is expressed, and how long it is expressed
25.4 Control of Gene Expression
• Gene Expression in Eukaryotes– Levels of Gene Control
1. Pretranscriptional control
2. Transcriptional control
3. Posttranscriptional control
4. Translational control
5. Posttranslational control
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functional protein
polypeptide chain
Posttranscriptional control
Transcriptional control
nuclear pore
intron exon
histones
nuclear envelope
Pretranscriptionalcontrol
primarymRNA
maturemRNA
Translationalcontrol
Posttranslationalcontrol
plasmamembrane
5´
3´
3´
5´
Levels of Gene Control in Eukaryotes
25.4 Control of Gene Expression
1. Pretranscriptional Control– Use DNA methylation and chromatin packing to keep
genes turned off– Heterochromatin: inactive genes located within darkly
staining portions of chromatin • Barr body• Tortoiseshell cats
– Euchromatin: loosely packed areas of active genes• Still needs to be “unpacked” before it can be transcribed• Chromatin remodeling complex pushes aside nucleosomes
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© Photodisc/Getty RF;
active X chromosome
inactive X
inactive X
active X chromosome
cell division Barr bodies
Coats oftortoiseshellcats havepatches oforange andblack.
allele fororange color
allele forblack color
Females have twoX chromosomes.
One X chromosome is inactivatedin each cell. Which one of the pairis inactivated is random.
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histone
nucleosome
Chromatinremodelingcomplex
DNA
X-Inactivation
25.4 Control of Gene Expression
2. Transcriptional Control– Transcription factors bind to promoter to form
transcription initiation complex– Transcription activators speed transcription
dramatically• Bind to enhancer
3. Posttranscriptional Control– Primary RNA processed
• Addition of poly-A tail and a guanine cap• Removal of introns and splicing of exons
– Different patterns of splicing can occur
25.4 Control of Gene Expression
4. Translational Control– Differences in the poly-A tails and/or guanine caps
may determine how long a mRNA is available for translation
– Specific hormones may also effect longevity of mRNA
5. Posttranslational Control– Some proteins must be activated after synthesis– Many proteins function only for a short time before
they are degraded or destroyed by the cell
25.5 Gene Mutations
• Permanent change in the sequence of bases in DNA
• Effect can range from none to complete inactivity of the protein
• Germ-line mutations can be passed to subsequent generations
• Some mutations can lead to cancer
25.5 Gene Mutations
• Three causes of mutations– Error in replication
• Proofreading by DNA polymerase usually takes care of this
– Mutagens• Radiation and certain organic chemicals• DNA repair enzymes constantly monitor and repair any
irregularities
– Transposons – jumping genes• Specific DNA sequences that move within and between
chromosomes• Sometimes alters neighboring gene expression• Likely all organisms (including humans) have them
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Normal gene
a.
b. c.
Mutated gene
transposon
cannotcode forpurple
pigment
codes forpurple
pigment
C: © Mondae Leigh Baker;
25.5 Gene Mutations
• Effect of Mutations on Protein Activity– Point mutations involve a change in a single
DNA nucleotide• Possible change in a specific amino acid• May have no effect• May produce an abnormal protein
– Sickle cell
• May produce an incomplete protein
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a.
No mutationC G T
His Leu Thr Pro Glu Glu
His Leu Thr Pro Glu Glu
His Leu Thr Pro Glu
His Leu Thr Pro Stop
A
A
A
(normal protein)His His
(incomplete protein)
Glu Stop
Glu Val
(abnormal protein)
Val
Val
Val
Val
Val
A C T G G A G GT C C CC TTGGA
C G TA C T G G GT C C CC TTGGAA
C G TA C T G G GT CC CCTGGAAG
C G TA C T G G GT C C CTGGAAG
3' 5'
T
b. Normal red blood cell c. Sickled red blood cell
© Stan Flegler/Visuals Unlimited;
25.5 Gene Mutations
• Frameshift Mutations– One or more nucleotides are either inserted or
deleted from DNA– Result can be a completely new sequence of
codons and a nonfunctional protein
THE CAT ATE THE RAT
THE ATA TET HER AT
25.5 Gene Mutations
• Nonfunctional Proteins– A single nonfunctioning protein can have a dramatic
effect on the phenotype, because enzymes are often a part of metabolic pathways
– PKU (phenylketonuria)• Phenylalanine can’t be broken down and builds up
causing mental impairment– Androgen insensitivity
• Faulty receptor makes cells unable to respond• Genetic male looks like a normal female
25.5 Gene Mutations
• Mutations can cause cancer– In the United States, the three deadliest forms
of cancer are lung cancer, colon and rectal cancer, and breast cancer
– Development of cancer involves a series of accumulating mutations that can be different for each type of cancer
– Carcinogenesis begins with the loss of tumor suppressor gene activity and/or the gain of oncogene activity
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receptorstimulating growth factor
cytoplasm
transcription factor
nucleus
oncogene
plasmamembrane
protein thatoverstimulatesthe cell cycle
signalingpathway
25.5 Gene Mutations
• Although cancers vary greatly, they usually follow a common multistep progression
• Most cancers begin as an abnormal cell growth that is benign, or not cancerous, and usually does not grow larger
• Growth may become malignant, meaning that it is cancerous and possesses the ability to spread
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epithelial cells 1 mutationCell (dark pink) acquires a mutation for repeated cell division.
New mutations arise, and one cell (green) has the ability to start a tumor.
The tumor is at itsplace of origin.One cell (purple)mutates further.
2 mutations
3 mutations tumor
lymphaticvessel
bloodvessel
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Cells have gained the ability to invade underlying tissues by producing a proteinase enzyme.
Cancer cells nowhave the ability toinvade lymphaticand blood vessels.
New metastatictumors are foundsome distancefrom the tumor.
invasivetumor
malignanttumor
distant tumor
lymphaticvessel
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epithelial cells 1 mutationCell (dark pink) acquires a mutation for repeated cell division.
New mutations arise, and one cell (green) has the ability to start a tumor.
The tumor is at itsplace of origin.One cell (purple)mutates further.
Cells have gained the ability to invade underlying tissues by producing a proteinase enzyme.
Cancer cells nowhave the ability toinvade lymphaticand blood vessels.
New metastatictumors are foundsome distancefrom the tumor.
2 mutations
3 mutations tumor
lymphaticvessel
bloodvessel
invasivetumor
malignanttumor
distant tumor
lymphaticvessel
Development of Cancer
25.5 Gene Mutations
• Characteristics of Cancer Cells– Cancer cells are genetically unstable– Cancer cells do not correctly regulate the cell
cycle– Cancer cells escape the signals for cell death– Cancer cells can survive and proliferate
elsewhere in the body