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David Sadava, David M. Hillis,
H. Craig Heller, May R. Berenbaum
La nuova
biologia.blu
Genetica, biologia molecolare ed
evoluzione S
DNA and Gene Expression
What Is the Evidence that the Gene Is DNA?
By the 1920s it was known that chromosomes
consisted of DNA and proteins.
A new dye that stained DNA provided evidence that
DNA is the genetic material.
• It was in the right place
• It varied among species
• It was present in the right amounts
What Is the Evidence that the Gene Is DNA?
Experimental evidence came from work on two strains
of Streptococcus pneumoniae.
A substance from cells of one strain (even when dead)
could produce a heritable change in the other strain.
What Is the Evidence that the Gene Is DNA?
To identify this substance,
Oswald Avery treated
samples to destroy
different molecules.
If DNA was destroyed, the
transforming activity was
lost.
There was no loss of
activity with destruction of
proteins or RNA.
What Is the Evidence that the Gene Is DNA?
Hershey-Chase experiment:
used bacteriophage T2 virus
to determine whether DNA,
or protein, is the genetic
material.
Part of the virus enters E. coli
cells and converts the cell
into a virus replication
machine.
What Is the Evidence that the Gene Is DNA?
Bacteriophage were grown
with either 35S to label the
proteins, or with 32P to
label the DNA.
After infection, bacterial
cells and viral remains
were separated—the
bacteria cells were labeled
with 32P, indicating that
DNA had entered the
cells.
What Is the Structure of DNA?
The structure of DNA was determined using many
lines of evidence.
One crucial piece came from X-ray diffraction.
Rosalind Franklin prepared crystallographs from DNA
samples.
Her images suggested a double-stranded helix with
10 nucleotides in each full turn.
What Is the Structure of DNA?
Chemical composition:
Biochemists knew that DNA is a polymer of
nucleotides.
Each nucleotide consists of deoxyribose, a phosphate
group, and a nitrogen-containing base.
The four different nucleotides differed only in the
bases:
•Purines: adenine (A), guanine (G)
•Pyrimidines: cytosine (C), thymine (T)
What Is the Structure of DNA?
Erwin Chargaff noticed that in all DNA, the amount of
purines is the same as the amount of pyrimidines.
Chargaff’s rule
What Is the Structure of DNA?
Francis Crick and James Watson used model
building, plus the physical and chemical evidence to
solve the structure of DNA.
They published their results in 1953.
What Is the Structure of DNA?
Four key features of DNA structure:
• It is a double-stranded helix
• It is right-handed
• It is antiparallel
• The outer edges of the bases are exposed in major
and minor grooves
What Is the Structure of DNA?
Antiparallel strands: direction of strand is determined
by the sugar–phosphate bonds.
Phosphate groups connect to the 3′ C of
one sugar, and the 5′ C of the next sugar.
Results in one chain with a free 5′
phosphate group—the 5′ end;
The other chain has is a free
3′ hydroxyl group—the 3′
end.
What Is the Structure of DNA?
The two chains are held together by:
1. Hydrogen bonding between bases –
complementary base pairing:
One purine (A or G) with one pyrimidine (T or C)
2. Van der Waals forces between adjacent bases
on the same strand.
What Is the Structure of DNA?
The double-helix structure is essential to DNA
function:
• Stores genetic information: with millions of
nucleotides, the base sequences store a huge
amount of information
• Susceptible to mutations
• Precisely replicated in cell division by
complementary base pairing
• Genetic information is expressed as the
phenotype—nucleotide sequence determines
sequence of amino acids in proteins
How Is DNA Replicated?
The mechanism of DNA replication is
semiconservative (each parent strand is a template;
new molecules have one old and one new strand).
Ingredients needed:
• Deoxyribonucleoside triphosphates dATP, dCTP,
dGTP, and dTTP (dNTPs, the monomers of DNA)
• DNA molecules to serve as template
• DNA polymerase enzyme
How Is DNA Replicated?
Two steps in DNA replication:
• Double helix is unwound, making two template
strands
• New nucleotides form complementary base pairs
with template DNA and are linked by
phosphodiester bonds
How Is DNA Replicated?
Nucleotides are added to the new strand at the 3′ end.
Formation of the phosphodiester linkage is a
condensation reaction.
Bonds linking the
phosphate groups
of the
triphosphate
nucleosides are
broken, releasing
energy that drives
the reaction.
How Is DNA Replicated?
DNA polymerase requires a primer, a short starter
strand—usually RNA.
The primer is complementary to the DNA template
and is synthesized by an enzyme called a primase.
DNA polymerase then adds
nucleotides to the 3′ end until
that section is complete, and
the primer is degraded.
How Is DNA Replicated?
At the replication fork DNA opens up like a zipper in
one direction.
The leading strand grows at its 3′ end as the fork
opens.
In the lagging strand the exposed 3′ end gets farther
from the fork, and an unreplicated gap forms.
How Is DNA Replicated?
Synthesis of the lagging strand occurs in small,
discontinuous stretches called Okazaki fragments.
The final phosphodiester linkage between fragments
is catalyzed by DNA ligase.
How Is DNA Replicated?
Eukaryote chromosomes have repetitive sequences at
the ends called telomeres.
In humans the sequence is TTAGGG, repeated about
2,500 times.
Continuously dividing cells have telomerase, which
catalyzes addition of lost telomeres.
Telomerase is expressed in
most cancer cells, and is
important in their ability to
keep dividing. It is a target
for anti-cancer drugs.
How Are Errors in DNA Repaired?
DNA polymerases initially make many mistakes, and
DNA can be damaged by chemicals, UV radiation,
and other threats.
Cells have three repair mechanisms:
proofreading, mismatch repair, excision repair.
How Does Information Flow from Genes to Proteins?
Gene expression occurs in two steps:
•Transcription: DNA sequence is copied to a
complementary RNA sequence
•Translation: RNA sequence is template for an amino
acid sequence
This model was proposed by Crick and Watson, and
called “The central dogma of molecular biology.”
How Does Information Flow from Genes to Proteins?
The central dogma suggested that information flows
from DNA to RNA to protein, which raised two
questions:
• How does genetic information get from the nucleus
to the cytoplasm?
• What is the relationship
between a DNA sequence
and an amino acid
sequence?
How Does Information Flow from Genes to Proteins?
Three kinds of RNA are involved in gene expression:
1.Messenger RNA (mRNA) and transcription— one
strand of DNA is copied to a complementary mRNA
strand. In eukaryotes, the mRNA moves to the
cytoplasm.
2.Ribosomal RNA (rRNA) and translation—
ribosomes are protein synthesis factories made up
of proteins and rRNA
3.Transfer RNA (tRNA)— can bind a specific amino
acid, and recognize specific sequences in mRNA.
How Is the Information Content in DNA Transcribed to Produce RNA?
Transcription occurs in three phases:
1. Initiation:
RNA polymerase binds to a DNA sequence called a
promoter.
Promoters tell the enzyme where to start and which
strand of DNA to transcribe.
The promoter has an
initiation site where
transcription begins.
How Is the Information Content in DNA Transcribed to Produce RNA?
2. Elongation:
RNA polymerase unwinds DNA about 10 base pairs at
a time; reads template in 3′ to 5′ direction.
The transcript is antiparallel to the DNA template
strand.
RNA polymerases do not proofread and correct
mistakes.
How Is the Information Content in DNA Transcribed to Produce RNA?
3. Termination:
Specified by a specific DNA sequence.
Mechanism in eukaryotes is not well understood.
In bacteria, the transcript forms a loop and falls away
from the DNA; or a helper protein binds to the
transcript and causes it to detach from the DNA.
How Is the Information Content in DNA Transcribed to Produce RNA?
The genetic code specifies which amino acids will be
used to build a protein.
Codon: a sequence of three bases, something like a
three-letter “word.”
Each codon specifies a particular amino acid.
How Is the Information Content in DNA Transcribed to Produce RNA?
AUG is the start codon.
Stop codons— UAA, UAG, and UGA.
The genetic code is nearly universal, redundant
(more codons than amino acids) but not ambiguous.
How Is RNA Translated into Proteins?
Transfer RNA (tRNA) links mRNA codons with
specific amino acids.
Each tRNA has three functions:
• It binds to a specific enzyme that attaches it to only
one amino acid: it is then “charged”
• Binds to mRNA at a triplet called the anticodon,
which is complementary to an mRNA codon
• Interacts with ribosomes
How Is RNA Translated into Proteins?
tRNAs are charged by aminoacyl-tRNA synthetases.
Each enzyme is specific for one amino acid and its
corresponding tRNA.
Translation occurs at a ribosome.
It holds mRNA and charged tRNAs in the correct
position to allow assembly of the polypeptide.
Ribosomes can make any type of protein, they can be
used over and over. Most cells have thousands of
them.
How Is RNA Translated into Proteins?
Ribosomes have two subunits, large and small, held
together non-covalently.
In eukaryotes, the large subunit has three different
molecules of ribosomal RNA (rRNA) and 49 different
proteins in a precise pattern.
The small subunit has one rRNA and 33 proteins.
How Is RNA Translated into Proteins?
A large subunit has three tRNA binding sites:
• A (aminoacyl tRNA) site binds with anticodon of
charged tRNA
• P (peptidyl tRNA) site where tRNA adds its amino
acid to the growing chain
• E (exit) site where tRNA sits before being released
from the ribosome
How Is RNA Translated into Proteins?
Translation occurs in three steps:
1. Initiation
An initiation complex forms—a
charged tRNA and small
ribosomal subunit, both bound to
mRNA.
How Is RNA Translated into Proteins?
2. Elongation
Another charged tRNA enters A site
and the large subunit catalyzes two
reactions:
• Bond between tRNA in P site and
its amino acid is broken
• Peptide bond forms between that
amino acid and the amino acid on
tRNA in the A site
How Is RNA Translated into Proteins?
3. Termination
Translation ends when a stop codon enters the A site.
Stop codons bind a protein release factor which
hydrolyzes bond between the polypeptide and the
tRNA in the P site.
The polypeptide then separates from the ribosome.
What Happens to Polypeptides after Translation?
After translation, polypeptides may move into an
organelle, or out of the cell.
They are often modified by the addition of new
chemical groups that affect their function.
Polypeptide emerges from the ribosome and folds
into its 3-D shape.
It may contain a signal sequence indicating
where in the cell it belongs.
Example: a nuclear localization signal (NLS) -Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-
What Happens to Polypeptides after Translation?
What Happens to Polypeptides after Translation?
Most polypeptides are modified after translation:
• Proteolysis: Polypeptide is cut by proteases, (e.g.,
signal sequence is removed)
• Glycosylation: Addition of sugars to form
glycoproteins. The sugars can act as signals; others
form membrane receptors
• Phosphorylation:
Addition of phosphate
groups catalyzed by
protein kinases
Adapted from
Life: The Science of Biology, Tenth Edition, Sinauer Associates, Sunderland, MA, 2014
Inc. All rights reserved