Transcript Chapter 14

Chapter 14
DNA Replication
Learning Objectives
• Diagram the process of eukaryotic vs. prokaryotic DNA
replication
• Describe the semiconservative process of DNA
replication
• Diagram the structure of DNA (ie what are based like?
How are they paired, where is the sugar backbone
located and its general overall shape)
• Name the 4 enzymes involved in DNA synthesis and
their functions
• Assess the importance of telomeres and telomerase
• Describe the process and importance of DNA
proofreading during replication
• List the function and components of histones
DNA
• Stores information in a double helix
• Structure was postulated by Watson and Crick,
based on Xray crystallography done by Rosalind
Franklin
• DNA molecule consists of two polynucleotide
chains twisted around each other into a righthanded double helix
• Each nucleotide of the chains consists of
– Deoxyribose
– A phosphate group
– A base (adenine, thymine, guanine, or cytosine)
Structure
• Deoxyribose sugars are linked by phosphate
groups to form a sugar– phosphate backbone
• Two strands are held together by base pairs
– Adenine–Thymine, Guanine–Cytosine
• Each full turn of double helix is 10 base pairs
5' end
Phosphate
Deoxyribose (a 5-carbon sugar)
Adenine
(A)
Guanine
(G)
Purines
(double-ring
structures)
Thymine
(T)
Cytosine
(C)
Pyrimidines
(single-ring
structures)
Hydroxyl group
3' end
Fig. 14-4, p. 281
2 nm
5' end
3' end
Distance between
each pair of bases =
0.34 nm
Each full twist of
the DNA double helix =
3.4 nm
5-carbon sugar
deoxyribose)
Nitrogenous
base (guanine)
Phosphate
group
3' end
Hydrogen
bond
5' end
Fig. 14-6, p. 283
DNA replication
• DNA polymerases are the primary enzymes of DNA
replication
• Helicases unwind DNA to expose template strands for
new DNA synthesis
• RNA primers provide the starting point for DNA
polymerase to begin synthesizing a new DNA chain
• One new DNA strand is synthesized continuously; the
other, discontinuously
Assembling Antiparallel Strands
• Meselson and Stahl showed that DNA
replication is semiconservative
– Two strands of parental DNA molecule unwind
– Each is a template for the synthesis of a
complementary copy
a. Semiconservative
replication
KEY
Parental
DNA
Replicated
DNA
1st
replication
2nd
replication
The two parental strands of DNA unwind, and each is
a template for synthesis of a new strand. After
replication has occurred, each double helix has one
old strand paired with one new strand.
Fig. 14-8a, p. 285
Enzymes of DNA Replication
• Helicase unwinds the DNA
• Primase synthesizes RNA primer (starting point for
nucleotide assembly by DNA polymerases)
• DNA polymerases assemble nucleotides into a chain,
remove primers, and fill resulting gaps
• DNA ligase closes remaining single-chain nicks
Telomerase
• Ends of eukaryotic chromosomes
• Short sequences repeated hundreds to
thousands of times
• Repeats protect against chromosome
shortening during replication
• Chromosome shortening is prevented in
some cell types which have a telomerase
enzyme (adds telomere repeats to
chromosome ends)
3' end of
template strand
1 3' end of DNA
template unwound
and ready for
replication.
2 Primer added and
new DNA assembled
from end of primer.
Primer
New DNA
3 Primer removed.
Gap left by
primer removal
Chromosome strand
shortened
Fig. 14-13, p. 291
Original end
of chromosome
1 Extra telomere
repeats added by
telomerase at 3’ end
of template strand
Added telomere repeats
2 Primer added
and gap filled in
Primer added to
chromosome end
Gap filled in
3 Primer removed;
original length is
restored
Primer removed
Chromosome strand
not shortened
Fig. 14-14, p. 291
DNA Synthesis
• Begins at sites that act as replication
origins
• Proceeds from the origins as two
replication forks moving in opposite
directions
Origin DNA double helix
Replication forks
Replication direction
Fig. 14-15, p. 292
Origin
Replication forks
DNA double helix
Fig. 14-19, p. 297
Proofreading
• If a replication error causes a base to be
mispaired, DNA polymerase reverses and
removes the most recently added base
• Proofreading depends on the ability of
DNA polymerases to reverse and remove
mismatched bases
• DNA repair corrects errors that escape
proofreading
Template strand
DNA polymerase
1 Enzyme continues activity
New strand
in the forward direction as
DNA 3’ polymerase as long as
the most recently added
nucleotide is correctly paired.
2 Enzyme adds a mispaired
nucleotide.
New strand
3 Enzyme reverses, acting
as a deoxyribonuclease to
remove the mispaired
nucleotide.
4 Enzyme resumes forward
activity as a DNA polymerase.
Fig. 14-16, p. 293
Template strand
Base-pair mismatch
1 Repair enzymes recognize
a mispaired base and break
one chain of the DNA at the
arrows.
New strand
2 The enzymes remove
several to many bases,
including the mismatched
base, leaving a gap in the
DNA.
3 The gap is filled in by a DNA
polymerase using the intact
template strand as a guide.
Nick left after
gap filled in
4 The nick left after gap filling
is sealed by DNA ligase to
complete the repair.
Fig. 14-17, p. 293
DNA Organization
in Eukaryotes and Prokaryotes
• Histones pack eukaryotic DNA at
successive levels of organization
• Many nonhistone proteins have key roles
in the regulation of gene expression
• DNA is organized more simply in
prokaryotes than in eukaryotes
Chromatin
• Distributed between:
– Euchromatin (loosely packed region, genes
active in RNA transcription)
– Heterochromatin (densely packed masses,
genes are inactive)
• Folds and packs to form thick, rodlike
chromosomes during nuclear division
The Bacterial Chromosome
• Closed, circular molecule of DNA packed
into nucleoid region of cell
• Replication begins from a single origin,
proceeds in both directions
• Plasmids (in many bacteria) replicate
independently of the host chromosome
Learning Objectives
• Diagram the process of eukaryotic vs. prokaryotic DNA
replication
• Describe the semiconservative process of DNA
replication
• Diagram the structure of DNA (ie what are bases like?
How are they paired, where is the sugar backbone
located and its general overall shape)
• Name the 4 enzymes involved in DNA synthesis and
their functions
• Assess the importance of telomeres and telomerase
• Describe the process and importance of DNA
proofreading during replication
• List the function of histones
Chapter 16: Gene regulation
• Diagram the lac operon transcription unit
• Compare and contrast the operon model
of tryptophan and lactose metabolism
• Compare and contrast prokaryotic and
eukaryotic gene regulation
Gene Expression Control
All somatic cells in an organism are
genetically identical
– Cells differentiate by gene expression
• Gene expression is collectively controlled
through transcriptional regulation
– Main control: Gene transcribed into mRNA
– Additional controls: Posttranscriptional,
translational and posttranslational
Prokaryotic Gene Expression
• Operon is the unit of transcription in
prokaryotes
• lac operon for lactose metabolism is
transcribed when an inducer inactivates a
repressor
• Transcription of the lac operon is also
controlled by a positive regulatory system
Operon: Unit of Transcription
• Prokaryotic gene expression reflects life history
– Rapid, reversable response to environment
• Operon: A cluster of prokaryotic genes and DNA
sequences involved in their regulation
– RNA polymerase binds at promoter for operon
– Many genes may be transcribed into one mRNA
– Cluster of genes is transscriptional unit
Operon: Unit of Transcription (2)
• Regulatory proteins bind at operator
– Regulatory protein coded by gene outside operon
• Repressor proteins prevent operon genes from being
expressed
• Activator proteins turn on expression of genes from
operon
lac Operon for Lactose
Metabolism
• Lactose metabolism in E. Coli requires
three genes lacZ, lacY and lacA
– lac operon contains all three genes and
regulatory sequences
• lac operon operator sequence is between
promoter and lacZ
Sequences that control the
expression of the operon
Regulatory gene
lacI
lac operon
Promoter Operator
lacZ
lacY
lacA
DNA
Binds RNA Binds Lac
polymerase repressor
Transcription
initiation site
Lac repressor
β-Galactosidase
Transcription
termination site
Permease Transacetylase
Fig. 16-2, p. 331
lac Operon for Lactose
Metabolism
• lac repressor stops lac operon expression
– Encoded by lacI, synthesized in active form
– Binds promoter, prevents transcription
• Allolactose made from lactose when it enters cell, lasts
as long as lactose available
– Inducer of lac operon by binding to lac repressor
– Inducible operon because inducer increases
expression
a. Lactose absent from medium
lac operon
lacI
Promoter Operator
lacZ
lacY
lacA
DNA
Transcription
blocked
mRNA
Lac
repressor
(active)
RNA polymerase
cannot bind
to promoter
When lactose is
absent from the
medium, the active
Lac repressor
binds to the
operator of the lac
operon, blocking
transcription.
Fig. 16-3a, p. 332
b. Lactose present in medium
lac operon
lacI
Promoter Operator
DNA
mRNA
Lac
repressor
(active)
RNA polymerase
binds and
transcribes operon
Binding site
for inducer
Allolactose
(inducer)
lacZ
lacY
lacA
Transcription
occurs
Inactive
repressor
Translation
Lactose
metabolism
enzymes
mRNA
When lactose is present in
the medium, some of it is
converted to the inducer
allolactose. Allolactose binds
to the Lac repressor,
inactivating it so that it
cannot bind to the operator.
This allows RNA polymerase
to bind to the promoter, and
transcription of the lac
operon occurs. Translation of
the mRNA produces the three
lactose metabolism enzymes.
Fig. 16-3b, p. 332
Positive Regulation of lac
Operon
• lac operon operates when lactose but not
glucose is present
– Glucose more efficient energy source than
lactose
• Catabolite Activator Protein (CAP) is an
activator that stimulates gene expression
– CAP activated by cAMP
– cAMP only abundant when glucose levels are
low
a. Lactose present; glucose low or absent
CAP site Promoter Operator
Transcription
occurs
lacZ
lacI
DNA
cAMP
CAP
mRNA
mRNA
RNA
polymerase
Translation
Active CAP
Lac
repressor
(active)
Allolactose
(inducer)
Lactose
metabolism
enzymes
Inactive
repressor
When lactose is
present and glucose
is low or absent,
cAMP levels are high.
cAMP binds to CAP,
activating it. Active
CAP binds to the CAP
site and recruits
RNA polymerase to
the promoter.
Transcription then
occurs.
Fig. 16-5a, p. 334
a. Lactose present; glucose low or absent
CAP site Promoter Operator
Transcription
occurs
lacZ
lacI
DNA
cAMP
CAP
mRNA
mRNA
RNA
polymerase
Active CAP
Lac
repressor
(active)
Allolactose
(inducer)
Translation
Lactose
metabolism
enzymes
Inactive
repressor
When lactose is
present and glucose
is low or absent,
cAMP levels are high.
cAMP binds to CAP,
activating it. Active
CAP binds to the CAP
site and recruits
RNA polymerase to
the promoter.
Transcription then
occurs.
Stepped Art
Fig. 16-5a, p. 334
b. Lactose present; glucose present
lacI
CAP site Promoter Operator
No
transcription
lacZ
DNA
RNA polymerase
binding site
mRNA
Inactive
CAP
RNA
polymerase
cannot bind
Lac
repressor
(active)
Allolactose
(inducer)
Inactive
repressor
When lactose is
present and glucose
is present, cAMP
levels are low. As a
result, CAP is inactive
and cannot bind to
the CAP site. RNA
polymerase then is
unable to bind to the
promoter, and no
transcription occurs.
Fig. 16-5b, p. 334
Summary of lac operon
• Turn off unless Lactose is present (lac I
protein active)
• Turn on if Lactose is present (Lac I binding
to allolactose; inactive)
• Turn on if Lactose is present (CAMP binds
to CAP to activate
• Turn off again if Lactose AND Glucose are
present (CAMP not available with glucose
present; cannot activate CAP)
Tryptophan metabolism
• No Tryp available, the cell makes it ownthe operon is turned “on”
• If tryp is available, the cell does not want
the enzymes for synthesis to be made, so
the cell turns it off
• This is negative regulation – turning off
rather than on
a. Tryptophan absent from medium
Regulatory RNA polymerase binds
and transcribes operon
gene
trpR
Promoter Operator trpE
trp operon
trpD
trpC
trpB
trpA
DNA
mRNA
Trp
repressor
(inactive)
Transcription
occurs
mRNA
Translation
Tryptophan
biosynthesis
enzymes
When tryptophan is
absent from the
medium, the Trp
repressor is inactive in
binding to the operator
and transcription
proceeds.
Fig. 16-4a, p. 333
b. Tryptophan present in medium
trp operon
trpR
Promoter Operator trpE
trpD
trpC
trpB
trpA
DNA
Transcription
blocked
mRNA
Trp
repressor
(inactive)
RNA polymerase
cannot bind
to promoter
Tryptophanbinding site Tryptophan
(corepressor)
Trp repressor
(active)
When tryptophan is
present in the
medium, the amino
acid binds to, and
activates, the Trp
repressor. The active
repressor binds to the
operator and blocks
transcription.
Fig. 16-4b, p. 333
Eukaryotic Transcription Regulation
• In eukaryotes, regulation of gene expression occurs at
several levels
• Chromatin structure plays an important role in whether a
gene is active or inactive
• Regulation of transcription initiation involves a gene’s
promoter and regulatory sites
• Methylation of DNA can control gene transcription
Regulation of Gene Expression
in Eukaryotes
• Gene expression in eukaryotes has more
regulatory points
– Chromatin has histones
– Different types of cells
– Nuclear envelope
• Three main areas of eukaryotic regulation
of gene expression
– Transcriptional, posttrascriptional and
posttranslational
Chapter 16: Gene regulation
• Diagram the lac operon transcription unit
• Compare and contrast the operon model
of tryptophan and lactose metabolism
• Compare and contrast prokaryotic and
eukaryotic gene regulation