Download DNA and Genetic Material

DNA and Genetic Material
Chapters 3 and 25
Yes, we are moving around a
little!
• Composed of 4 nucleotide
bases, 5 carbon sugar and
phosphate.
• Base pair = rungs of a
ladder.
• Edges = sugar-phosphate
backbone.
• Double Helix
• Anti-Parallel
The bases
• Chargaff’s Rules
• A=T
• G=C
• led to suggestion of a
double helix structure
for DNA
The Bases
• Adenine (A) always base pairs with thymine (T)
• Guanine (G) always base pairs with Cytosine (C)
The Bases
• The C#T pairing on the left suffers from carbonyl dipole
repulsion, as well as steric crowding of the oxygens. The
G#A pairing on the right is also destabilized by steric
crowding (circled hydrogens).
DNA Replication
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Adenine (A) always base pairs with thymine (T)
Guanine (G) always base pairs with Cytosine (C)
ALL Down to HYDROGEN Bonding
Requires steps:
– H bonds break as enzymes unwind molecule
– New nucleotides (always in nucleus) fit into place
beside old strand in a process called
Complementary Base Pairing.
– New nucleotides joined together by enzyme called
DNA Polymerase
DNA Replication
• Each new double helix is composed of an old
(parental) strand and a new (daughter) strand.
• As each strand acts as a template, process is called
Semi-conservative Replication.
• Replication errors can occur. Cell has repair
enzymes that usually fix problem. An error that
persists is a mutation.
• This is permanent, and alters the phenotype.
DNA replication
•If only is was that
simple……….
DNA replication
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Origins of replication
The replication fork
Leading strand
Lagging strand
Dynamics at the replication fork
Regulation of replication
Origins of replication
• Targeted by proteins that separate the two
strands and initiate DNA synthesis.
• Contain DNA sequences recognized by replication
initiator proteins
• These initiator proteins recruit other proteins to
separate the two strands and initiate replication
forks
Origins of replication
• Initiator proteins recruit other
proteins to separate the DNA
strands at the origin, forming a
bubble.
• Tend to be "AT-rich" to assist
this process
• A-T base pairs have two
hydrogen bonds strands rich in
these nucleotides are generally
easier to separate due the
positive relationship between the
number of hydrogen bonds and
the difficulty of breaking these
bonds.
The replication fork
• structure that forms within the nucleus during DNA replication
• created by helicases : break H bonds between bases. The resulting
structure has two branching "prongs", each one made up of a single
strand of DNA.
Leading & Lagging strands
• The leading strand template is oriented in a 3' to 5'
manner.
• All DNA synthesis occurs 5'-3'. The original DNA strand
must be read 3'-5' to produce a 5'-3' daughter strand.
Leading & Lagging strands
• The lagging strand template is the coding strand of the DNA double
helix that is oriented in a 5' to 3' manner .
• The newly made lagging strand still is synthesized 5'-3'.
•
WHAT?
Leading & Lagging strands
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The DNA is oriented in a manner that does not allow continual synthesis, only small
sections can be read at a time.
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An RNA primer is placed on the DNA strand 3' to the origin of replication. Just as
before, DNA Polymerase reads 3'-5' on the original DNA to produce a 5'-3' daughter
strand.
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Polymerase reaches the origin of replication and stops replication until a new RNA
primer is placed 3' to the last RNA primer.
Okazaki fragments
•
Fragments of DNA produced on the lagging strand.
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The orientation of the original DNA on the lagging strand prevents continual
synthesis. As a result, replication of the lagging strand is more complicated than
of the leading strand.
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In eukaryotes, primase is intrinsic to DNA polymerase III which also lengthens
the primed segments, forming Okazaki fragments. Primer removal in eukaryotes
is also performed by this enzyme.
Dynamics at the replication fork
• As helicase unwinds DNA at the replication fork, the DNA
ahead is forced to rotate.
• Results in a build-up of twists in the DNA ahead. This buildup would form a resistance that would eventually halt the
progress of the replication fork.
• DNA topoisomerases are enzymes that solve these physical
problems in the coiling of DNA.
• Topoisomerase I cuts a single backbone on the DNA,
enabling the strands to swivel around each other to remove
the build-up of twists.
Dynamics at the replication fork
• Topoisomerase II cuts both backbones, enabling one
double-stranded DNA to pass through another, thereby
removing knots and entanglements that can form within and
between DNA molecules.
• Bare single-stranded DNA has a tendency to fold back upon
itself and form secondary structures; these structures can
interfere with the movement of DNA polymerase.
• To prevent this, single-strand binding proteins bind to the
DNA until a second strand is synthesized, preventing
secondary structure formation
Summary
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Topoisomerase: This enzyme initiates
unwinding of the double helix by cutting
one of the strands.
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Helicase: This enzyme assists the
unwinding. Note that many hydrogen
bonds must be broken if the strands are
to be separated..
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SSB: A single-strand binding-protein
stabilizes the separated strands, and
prevents them from recombining, so that
the polymerization chemistry can function
on the individual strands.
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DNA Polymerase: This family of enzymes
link together nucleotide triphosphate
monomers as they hydrogen bond to
complementary bases. These enzymes also
check for errors (roughly ten per billion),
and make corrections.
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Ligase: Small unattached DNA segments
on a strand are united by this enzyme.
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• mvgmgt
Central Dogma of Molecular
Biology
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DNA holds the code
DNA makes RNA
RNA makes Protein
DNA to DNA is called REPLICATION
DNA to RNA is called TRANSCRIPTION
RNA to Protein is called TRANSLATION
Central Dogma of Molecular
Biology
Summary of protein synthesis
• Proteins:
• Chains of Amino Acids
• Three nucleotide base pairs
code for one amino acid.
• Proteins are formed from
RNA
• The nucleotide code must be
translated into an amino acid
code.
Occurs in the cytoplasm or on
Rough ER
RNA
• Formed from 4
nucleotides, 5 carbon
sugar, phosphate.
• Uracil is used in RNA.
– It replaces Thymine
• The 5 carbon sugar has
an extra oxygen.
• RNA is single stranded.
Translation
• Translation requires:
– Amino acids
– Transfer RNA: (tRNA) Appropriate to its time,
transfers AAs to ribosomes. The AA’s join in
cytoplasm to form proteins. 20 types. Loop structure
– Ribosomal RNA: (rRNA) Joins with proteins made in
cytoplasm to form the subunits of ribosomes. Linear
molecule.
– Messenger RNA: (mRNA) Carries genetic material
from DNA to ribosomes in cytoplasm. Linear
molecule.
Translation
• Initiation—
– mRNA binds to smaller of ribosome subunits, then,
small subunit binds to big subunit.
– AUG start codon--complex assembles
• Elongation—
– add AAs one at a time to form chain.
– Incoming tRNA receives AA’s from outgoing tRNA.
Ribosome moves to allow this to continue
• Termintion—
Stop codon--complex falls apart
Translation
• Translation requires:
– Amino acids
– Transfer RNA: (tRNA) Appropriate to its time,
transfers AAs to ribosomes. The AA’s join in
cytoplasm to form proteins. 20 types. Loop structure
– Ribosomal RNA: (rRNA) Joins with proteins made in
cytoplasm to form the subunits of ribosomes. Linear
molecule.
– Messenger RNA: (mRNA) Carries genetic material
from DNA to ribosomes in cytoplasm. Linear
molecule.
Gene expression in bacteria
• Escherichia coli (E. coli); is
a Gram negative rodshaped bacterium that is
commonly found in the
lower intestine of warmblooded organisms.
• Part of the normal flora of
the gut, and can benefit their
hosts by producing vitamin K2, and
by preventing the establishment
of pathogenic bacteria within the
intestine
The LAC operon
• Jacob and Monod
• First scientists to elucidate a
transcriptionally regulated
system. They worked on
the lactose metabolism system
in E. Coli.
• When the bacterium is in an
environment that contains lactose
it should turn on the enzymes
that are required for lactose
degradation.
The LAC operon
• A bacterium's prime source
of food is glucose, since it
does not have to be modified
to enter the respiratory
pathway.
• So if both glucose and
lactose are around, the
bacterium wants to turn off
lactose metabolism in favor
of glucose metabolism.
• There are sites upstream of
the Lac genes that respond
to glucose concentration.
The LAC operon
• beta-galactosidase:
• This enzyme hydrolyzes the bond between
the two sugars, glucose and galactose.
• It is coded for by the gene LacZ.
• Lactose Permease:
• This enzyme spans the cell membrane and
brings lactose into the cell from the
outside environment. The membrane is
otherwise essentially impermeable to
lactose. It is coded for by the gene
LacY.
• Thiogalactoside transacetylase:
• The function of this enzyme is not known.
It is coded for by the gene LacA.
The LAC operon
• Operator (LacO)
– binding site for repressor
• Promoter (LacP)
– binding site for RNA polymerase
• Repressor (LacI)
– gene encoding lac repressor protein
– Binds to DNA at operator and blocks
binding of RNA polymerase at
promoter
• Pi
– promoter for LacI
• CAP
– binding site for cAMP/CAP complex
The LAC operon
• LacZ, Y, and A appear adjacent to
each other on the E. Coli genome.
• Preceded by a region which is
responsible for the regulation of
the lactose metabolic genes.
• It would seem that the cell would
want to turn these genes on when
there is lactose around and off
when lactose is absent.
• The story is more complicated
than that!
The LAC operon
• REMEMBER!!!!
• A bacterium's prime source
of food is glucose, since it
does not have to be modified
to enter the respiratory
pathway.
• So if both glucose and
lactose are around, the
bacterium wants to turn off
lactose metabolism in favor
of glucose metabolism.
• There are sites upstream of
the Lac genes that respond
to glucose concentration.
The LAC operon
• When lactose is present, it
acts as an inducer of the
operon.
• Enters the cell and binds to
the Lac repressor, inducing a
conformational change that
allows the repressor to fall
off the DNA.
• Now the RNA polymerase is
free to move along the DNA
and RNA can be made from
the three genes.
• Lactose can now be
metabolized.
The LAC operon
• When the inducer (lactose) is removed:
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Repressor returns to its original
conformation and binds to the DNA, so
that RNA polymerase can no longer get
past the promoter. No RNA and no protein
is made.
• RNA polymerase can still bind to the
promoter though it is unable to move past
it.
• When the cell is ready to use the operon,
RNA polymerase is already there and
waiting to begin transcription; the
promoter doesn't have to wait for the
enzyme to bind.
• We could say that the operon
is primed for transcription upon the
addition of lactose.
The LAC operon
• When levels of glucose (a catabolite) in
the cell are high, a cyclic AMP is
inhibited from forming.
• glucose levels drop, more cAMP forms.
• cAMP binds to a protein called CAP
(catabolite activator protein), which is
then activated to bind to the CAP
binding site.
• This activates transcription, perhaps
by increasing the affinity of the site
for RNA polymerase.
• This phenomenon is called catabolite
repression, a misnomer since it
involves activation, but understandable
since it seemed that the presence of
glucose repressed all the other sugar
metabolism operons.
Genetic engineering
Genetic engineering
• The direct alteration of a genotype
– Human genes can be inserted into human cells
for therapeutic purposes
– Genes can be moved from one species to
another
• Moving genes from human to human or
between species requires the use of special
enzymes known as restriction enzymes.
– These cut DNA at very specific sites
– They restrict DNA from another species –
isolated from bacteria.
Genetic engineering
• Transferred DNA is denatured to give ssDNA
• The probe will bind to gene of interest by
Complementary base-pairing - A with T and G with C
Genetically modified crops
• Agrobacterium method
– Uses the natural infection mechanism of
a plant pathogen
– Agrobacterium tumefaciens naturally
infects the wound sites in
dicotyledonous plant causing the
formation of the crown gall tumors.
– Capable to transfer a particular DNA
segment (T-DNA) of the tumor-inducing
(Ti) plasmid into the nucleus of infected
cells where it is integrated fully into
the host genome and transcribed,
causing the crown gall disease.
• So the pathogen inserts the new DNA with
great success!!!
Genetically modified crops
• The vir region on the plasmid inserts DNA between
the T-region into plant nuclear genome
• Insert gene of interest and marker in the T-region
by restriction enzymes – the pathogen will then
“infect” the plant material
• Works fantastically well with all dicot plant species
– tomatoes, potatoes, cucumbers, etc
– Does not work as well with monocot plant species - corn
• As Agrobacterium tumefaciens do not naturally
infect monocots
Genetically
modified
Figure
11.21 crops
•So to modify a plant:
•Need to know the DNA sequence of the gene of interest
•Need to put an easily identifiable maker gene near or next
to the gene of interest
•Have to insert both of these into the plant nuclear genome
•Good screen process to find successful insertion
•Clone the genetically altered plant
Genetically modified crops
• Can alter nutritional content
– Potatoes with 21-22% more starch
• Resistance to pathogens
– Less damage to crops – better total yield – lower retail
cost
• Herbicide-resistant plants
– Spraying the fields only kills weeds
• Longer shelf-lives
– More attractive to buy in bulk
Genetically modified crops
• Issues:
• Destroying ecosystems – tomatoes are now
growing in the artic tundra with fish antifreeze
in them!
• Destroying ecosystems – will the toxin now
being produced by pest-resistance stains kill
“friendly” insects such as butterflies.
• Altering nature – should we be swapping genes
between species?
Genetically modified crops
• Issues:
• Vegetarians – what about those tomatoes?
• Religious dietary laws – anything from a pig?
• Cross-pollination – producing a super-weed
• Human health – what of the antibiotic marker
gene?