Transcript Operating genetic material

The Living World
Fourth Edition
GEORGE B. JOHNSON
9
How Genes Work
PowerPoint® Lectures prepared by Johnny El-Rady
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9.1 The Griffith Experiment
Mendel’s work left a key question unanswered:
What is a gene?
The work of Sutton and Morgan established that
genes reside on chromosomes
But chromosomes contain proteins and DNA
So which one is the hereditary material
Several experiments ultimately revealed the
nature of the genetic material
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9.1 The Griffith Experiment
In 1928, Frederick Griffith discovered transformation
while working on Streptococcus pneumoniae
The bacterium exists in two strains
S
Forms smooth colonies in a culture dish
Cells produce a polysaccharide coat and can cause
disease
R
Forms rough colonies in a culture dish
Cells do not produce a polysaccharide coat and are
therefore harmless
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Fig. 9.1 How Griffith discovered transformation
Thus, the dead S bacteria
somehow “transformed” the live R
bacteria into live S bacteria
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9.2 The Avery and Hershey-Chase
Experiments
Two key experiments that demonstrated conclusively
that DNA, and not protein, is the hereditary material
Oswald Avery and his coworkers Colin MacLeod and
Maclyn McCarty published their results in 1944
Alfred Hershey and Martha Chase published their
results in 1952
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The Avery Experiments
Avery and his colleagues prepared the same mixture
of dead S and live R bacteria as Griffith did
They then subjected it to various experiments
All of the experiments revealed that the properties of
the transforming principle resembled those of DNA
1.
2.
3.
4.
Same chemistry and physical properties as DNA
Not affected by lipid and protein extraction
Not destroyed by protein- or RNA-digesting enzymes
Destroyed by DNA-digesting enzymes
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The Hershey-Chase Experiment
Viruses that infect bacteria have a simple structure
DNA core surrounded by a protein coat
Hershey and Chase used two different radioactive
isotopes to label the protein and DNA
Incubation of the labeled viruses with host bacteria
revealed that only the DNA entered the cell
Therefore, DNA is the genetic material
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Fig. 9.2 The
Hershey-Chase
Experiment
Thus, viral DNA
directs the
production of
new viruses
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9.3 Discovering the Structure of DNA
DNA is made up of nucleotides
Each nucleotide has a central sugar, a
phosphate group and an organic base
The bases are of two main types
Purines – Large bases
Adenine (A) and Guanine (G)
Pyrimidines – Small bases
Cytosine (C) and Thymine (T)
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Fig. 9.3 The four nucleotide subunits that make up DNA
Nitrogenous
base
5-C sugar
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Erwin Chargaff made key DNA observations that
became known as Chargaff’s rule
Purines = Pyrimidines
Rosalind Franklin’s
X-ray diffraction
experiments
revealed that DNA
had the shape of a
coiled spring or helix
A = T and C = G
Fig. 9.4
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Rosalind
Franklin
(1920-1958)
In 1953, James Watson and Francis Crick deduced
that DNA was a double helix
They came to their conclusion using Tinkertoy
models and the research of Chargaff and Franklin
Fig. 9.4
James Watson
(1928)
Francis Crick
(1916-2004)
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Fig. 9.4 The DNA
double helix
Dimensions
suggested by
X-ray diffraction
The two
possible
basepairs
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9.4 How the DNA Molecule Replicates
The two DNA strands are held together by weak
hydrogen bonds between complementary base pairs
A and T
C and G
If the sequence on one strand is
The other’s sequence must be
ATACGCAT
TATGCGTA
Each chain is a complementary mirror image of the
other
So either can be used as template to reconstruct the other
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There are 3 possible methods for
DNA replication
Fig. 9.5
Daughter DNAs
contain one old
and one new
strand
Original DNA
molecule is
preserved
Old and new
DNA are
dispersed in
daughter
molecules
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These three mechanisms were tested in 1958 by
Matthew Meselson and Franklin Stahl
Fig. 9.6
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Thus, DNA replication
is semi-conservative
Fig. 9.6
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How DNA Copies Itself
The process of DNA replication can be summarized
as such
The enzyme helicase first unwinds the double
helix
The enzyme primase puts down a short piece of
RNA termed the primer
DNA polymerase reads along each naked single
strand adding the complementary nucleotide
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Fig. 9.7 How nucleotides are added in DNA replication
Template strand
New strand
HO 3’
HO
5’
C
Sugarphosphate
backbone
Template strand
P
O
P
T
A
T
P
O
A
P
O
O
P
T
A
T
A
P
P
DNA polymerase
O
O
O
O
P
P
C
G
C
P
O
O
G
P
O
O
P
P
A
3’
OH
A
O
A
T
O
O
T
P
5’
P
G
O
P
P
5’
C
O
O
O
O
3’
P
G
New strand
P
P
P
Pyrophosphate
P
P
O
A
O
OH
P
5’
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3’
OH
DNA polymerase can only build a strand of DNA in
one direction
The leading strand is made continuously from one primer
The lagging strand is assembled in segments created
from many primers
Fig. 9.8
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RNA primers are removed and replaced with DNA
Ligase joins the ends of newly-synthesized DNA
Fig. 9.9
Mechanisms exist for DNA proofreading and repair
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9.5 Transcription
The path of genetic information is often called the
central dogma
DNA
RNA
Protein
A cell uses three kinds of RNA to make proteins
Messenger RNA (mRNA)
Transfer RNA (tRNA)
Ribosomal RNA (rRNA)
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9.5 Transcription
Gene expression is the use of information in DNA to
direct the production of proteins
It occurs in two stages
Fig. 9.10
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9.5 Transcription
The transcriber is
RNA polymerase
It binds to one DNA
strand at a site
called the promoter
It then moves along
the DNA pairing
complementary
nucleotides
It disengages at a
stop signal
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Fig. 9.11
9.6 Translation
Translation converts the order of the nucleotides of
a gene into the order of amino acids in a protein
The rules that govern translation are called the
genetic code
mRNAs are the “blueprint” copies of nuclear genes
mRNAs are “read” by a ribosome in threenucleotide units, termed codons
Each three-nucleotide sequence codes for an
amino acid or stop signal
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Fig. 9.12
The genetic code is (almost) universal
Only a few exceptions have been found
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Ribosomes
The protein-making factories of cells
They use mRNA to direct the assembly of a protein
A ribosome is
made up of two
subunits
Each of which
is composed
of proteins
and rRNA
Fig. 9.13
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Sites play key
roles in
translation
Transfer RNA
tRNAs bring amino
acids to the ribosome
They have two
business ends
Anticodon which is
complementary to
the codon on
mRNA
3’–OH end to
which the amino
acid attaches
Hydrogen
bonding causes
hairpin loops
3-D shape
Fig. 9.14
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Making the Protein
mRNA binds to the
small ribosomal
subunit
The large subunit
joins the complex,
forming the
complete ribosome
mRNA threads
through the
ribosome producing
the polypeptide
Fig. 9.16
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Fig. 9.15 How translation works
The process continues until a stop codon enters the A site
The ribosome complex falls apart and the protein is released
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9.7 Architecture of the Gene
In eukaryotes, genes are fragmented
They are composed of
Exons – Sequences that code for amino acids
Introns – Sequences that don’t
Eukaryotic cells transcribe the entire gene,
producing a primary RNA transcript
This transcript is then heavily processed to
produce the mature mRNA transcript
This leaves the nucleus for the cytoplasm
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Fig. 9.17 Processing eukaryotic mRNA
Protect from
degradation
and facilitate
translation
Different combinations of exons can generate different
polypeptides via alternative splicing
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6. The polypeptide chain
grows until the protetin is
completed.
7. Phosphorylation or other
chemical modifications can
alter the activity of a protein
after it is translated.
Amino
acid
Completed
polypeptide
tRNA
5’
Ribosome moves
toward 3’ end
Cytoplasm
Fig. 9.18 How
protein synthesis
works in
eukaryotes
Ribosome
5. tRNAs bring their amino
acids in at the A site of the
ribosome. Peptide bonds
form between amino acids at
the P site, and tRNAs exit the
ribosome from the E site.
4. tRNA molecules
become attached to
specific amino acids
with the help of
activating enzymes.
Amino acids are
brought to the
ribosome in the order
dictated by the mRNA.
DNA
Nuclear
membrane
3’
3’
RNA
polymerase
1. In the cell nucleus, RNA
polymerase transcribes
RNA from DNA
3’
Poly-A
tail
5’
5’
5’
3’
Primary
RNA transcript
Exons
Nuclear
pore
5’
Cap
Large
ribosomal
subunit
mRNA
Poly-A
tail
Introns
3’
Cap
Small
ribosomal
subunit
mRNA
2. Introns are excised from the RNA
transcript, and the remaining exons are
spliced together, producing mRNA
3. mRNA is transported out of the
nucleus. In the cytoplasm, ribosomal
subunits bind to the mRNA
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9.7 Architecture of the Gene
Most eukaryotic genes exist in multiple copies
Clusters of almost identical sequences called
multigene families
As few as three and as many as several
hundred genes
Transposable sequences or transposons are DNA
sequences that can move about in the genome
They are repeated thousands of times, scattered
randomly about the chromosomes
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9.8 Turning Genes Off and On
Genes are typically controlled at the level of
transcription
In prokaryotes, proteins either block or allow the
RNA polymerase access to the promoter
Repressors block the promoter
Activators make the promoter more accessible
Most genes are turned off except when needed
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The lac Operon
An operon is a segment of DNA that contains a
cluster of genes that are transcribed as a unit
The lac operon contains
Three structural genes
Encode enzymes involved in lactose metabolism
Two adjacent DNA elements
Promoter
Site where RNA polymerase binds
Operator
Site where the lac repressor binds
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The lac Operon
In the absence of lactose, the lac repressor binds to
the operator
RNA polymerase cannot access the promoter
Therefore, the lac operon is shut down
Fig. 9.19
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The lac Operon
In the presence of lactose, a metabolite of lactose
called allolactose binds to the repressor
This induces a change in the shape of the
repressor which makes it fall off the operator
RNA polymerase can now bind to the
promoter
Transcription of the lac operon is ON
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Fig. 9.19
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The lac Operon
What if the cell encounters lactose, and it already
has glucose?
The bacterial cell actually prefers glucose!
The lac operon is also regulated by an activator
The activator is a protein called CAP
It binds to the CAP-binding site and gives the
RNA polymerase more access to the promoter
However, a “low glucose” signal molecule has to
bind to CAP before CAP can bind to the DNA
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Fig. 9.20 Activators
and repressors of
the lac operon
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Enhancers
DNA sequences that make the promoters of genes
more accessible to many regulatory proteins at the
same time
Usually located
far away from
the gene they
regulate
Common in
eukaryotes; rare
in prokaryotes
Fig. 9.21
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9.9 Mutation
The genetic material can
be altered in two ways
Recombination
Change in the
positioning of the
genetic material
Mutation
Change in the
content of the
genetic material
Bithorax mutant
Fig. 9.22
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9.9 Mutation
Mutation and recombination provide the raw material
for evolution
Evolution can be viewed as the selection of particular
combinations of alleles from a pool of alternatives
The rate of evolution is ultimately limited by the
rate at which these alternatives are generated
Mutations in germ-line tissues can be inherited
Mutations in somatic tissues are not inherited
They can be passed from one cell to all its
descendants
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Kinds of Mutation
Mutations are caused in one of two ways
Errors in DNA replication
Mispairing of bases by DNA polymerase
Mutagens
Agents that damage DNA
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Kinds of Mutation
The sequence of DNA can be altered in one of two
main ways
Point mutations
Alteration of one or a few bases
Base substitutions, insertion or deletion
Frame-shift mutations
Insertions or deletions that throw off the
reading frame
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Fig. 9.23
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Kinds of Mutation
The position of genes can be altered in one of two
main ways
Transposition
Movement of genes from one part of the
genome to another
Occurs in both eukaryotes and prokaryotes
Chromosomal rearrangements
Changes in position and/or number of large
segments of chromosomes in eukaryotes
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Mutation, Smoking and Lung Cancer
Agents that cause cancer are called carcinogens
These are typically mutagens
The hypothesis that chemicals cause cancer was
first advanced in the 18th century
Many investigations since then have determined
that chemicals can cause cancer in both animals
and humans
For example, tars and other chemicals in
cigarette smoke can cause cancer of the lung
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