Transcript sequence of amino acids

Symptoms of cystic fibrosis
Salty sweat due to
altered salt secretion
in sweat ducts
Mucus-clogged
Airways;
Severe Respiratory
infections
Lung
Pancreas
Infertility in
males due
to clogged
sex ducts
Testis
Cell lining ducts
of the body
Cystic Fibrosis
• A single faulty protein is connected to the symptoms
• In 1989 the gene was mapped to chromosome #7
Our Goals
• To determine the connection between the symptoms
associated with cystic fibrosis and DNA – How?
• Learn how DNA replicates
• Understand the genetic code and how the instructions in
a gene are used to make a protein
Central Dogma of Biology
DNA and RNA: Polymers of Nucleotides
• Nucleotide: base + sugar + phosphate
Fig 10.2
T
A single strand
of DNA
A
Sugarphosphate
backbone
C
G
DNA vs. RNA Nucleotides
• Four nucleotides are found in DNA
– Differ in their nitrogenous bases
• Adenine (A), Thymine (T), Cytosine (C), Guanine
(G)
– Sugar:
• Four nucleotides are found in RNA
– Uracil instead of Thymine
• Adenine (A), Uracil (U), Cytosine (C), Guanine (G)
– Sugar:
DNA is like a rope ladder twisted into a spiral
DNA Structure
• Consists of 2
strands joined
together by weak
hydrogen bonds
• Rungs of the
ladder are
hydrogen bonded
N-bases
Fig 10.4
Twist
Base pairing in DNA:
Figure 10.5
What is a gene?
•
The kind of proteins an organism makes helps to
determine it’s phenotype
The order of bases in a gene determines......
•
The sequence of amino acids in the protein it codes
for, which determines......
•
the organism's phenotype—the physical and
biochemical characteristics of an organism.
DNA replication
Questions to answer
1. When during the cell cycle does it occur?
2. What do we start with and end with?
3. Where does it occur in a cell?
4. What’s needed for it to occur?
5. What is the sequence of events?
6. Why is it said to be semi-conservative?
7. What does proofreading mean?
8. What does the proofreading?
DNA
Replication
Fig 10.6
1
2
3
4
1. Parent molecule has 2 complementary strands of DNA
2. Enzymes initiate the breaking of the H-bonds, separating
the double helix
3. Free nucleotides base – pair to parent strands (A-T, G-C)
using DNA polymerase enzyme
4. Each “daughter” strand consists of one parental strand
and one new strand –
DNA can be damaged – eg. by ultraviolet light
• The enzymes (e.g. DNA polymerase) can repair the
damage:
– Is the damage always repaired? Consequences?
The Flow of
Genetic
Information:
DNA to RNA to
Protein
• Transcription:
Fig 10.9
• Translation:
ribosomes
translate mRNA
into protein—a
chain of amino
acids
CF phenotype
• Genes
determine
which
proteins a
cell can
make
• Proteins
control
phenotype
• e.g. CFTR
Gene codes
for CFTR
protein
CFTR Protein: The cystic fibrosis transmembrane regulator protein
Cytoplasm of cell
lining duct or lungs
CFTR Protein
Chloride ions
CFTR Protein
Cell
membrane
• Pumps
chloride ions
(salt) out of
cells lining
ducts of the
lungs
• What are the
consequences
when CFTR
doesn’t work?
Inside of duct
or
Air sac in lungs
The order of bases in a gene
determines the order of amino
acids in the protein it codes for
Fig 10.10
Why are proteins so important?
1. Enzymes: catalysts for nearly all chemical reactions in
cells; Determine what cells can make and digest
2. Structural components:
3. Receptors on cell surface
4. Hormones: e.g. insulin, growth hormone, prolactin
5. Transport: e.g. hemoglobin, spindle fibers
6. Immune system: antibodies
Transcription: copying DNA into RNA
Questions to answer:
1. What do we start with and end with?
2. Where does transcription occur? When?
3. What is needed for transcription to occur?
4. What is the sequence of events?
An RNA Nucleotide
Phosphate
Base
(Uracil, U)
Sugar:
ribose
This oxygen is
absent in
deoxyribose
RNA
• Base – pairing:
• Single stranded
• Sugar = ribose (one more oxygen than
deoxyribose)
Stages of
Transcription
Fig 10.13b
Fig 10.13a
Transcription
1. Initiation - RNA polymerase enzyme binds to
the promotor (section of DNA indicating “start
of a gene”)
2. Elongation – RNA polymerase catalyzes base
pairing on the template strand (U-A, G-C)
3. Termination – RNA polymerase reaches the
“stop” sequence and the new mRNA is released.
4. mRNA processing – non-coding regions of the
mRNA are removed and the mRNA leaves the
nucleus.
Fig. 10.14 Step
4 mRNA
processing
Translation: Ribosomes reading mRNA to
produce a polypeptide
Questions to answer
1.
2.
3.
4.
5.
What do we start with and end with?
Where does translation occur?
What is needed for translation to occur?
What is the sequence of events?
What are the roles of mRNA, ribosomes, start codon,
tRNA, anticodons, stop codon?
How do
ribosomes read
the code?
The genetic code
Fig 10.11
Codon = 3 letter
section of mRNA
that codes for
one amino acid
Transfer RNA: tRNA
tRNA
• Matches amino
acids with
codons in
mRNA using
anticodons
Fig 10.15
A portion of an mRNA molecule attached to a tRNA
Codon on mRNA
mRNA
Each Codon
specifies a specific
tRNA—amino acid
complex
Amino acid
Stages of translation
1. Initiation – mRNA start codon binds to tRNA
anticodon; Ribosome binds to both
2. Elongation
• tRNA brings specific AAs to the ribosome
as mRNA passes through the ribosome
(codon – anticodon recognition)
3. Termination – Ribosome reads an mRNA stop codon
(no tRNA with anticodon). mRNA and protein detach
from the ribosome
Explaining the symptoms of CF
1. Mucus build-up in the lungs
– Lung infections (e.g. pneumonia)
2. Male sterility (blocked vas deferens)
3. Salty sweat
4. Trouble digesting food (blocked
pancreatic duct)
Explaining the symptoms of CF
•
In CF, the faulty CFTR protein never makes it
to cell membrane
Understanding Cystic Fibrosis at the Cellular Level
How does CFTR protein get from where it’s produced to
its home in the cell membrane?
1. Where is the CFTR protein produced?
2. Where does it go for modification?
 How does it get there?
3. How does the modified CFTR protein get to the
plasma membrane?
4. The defective CFTR protein is recognized at the ER
as defective
 Where is the defective CFTR protein sent?
CF symptoms may be mild or severe
CFTR
Gene
Several hundred different mutations are associated with CF
What’s a Mutation?
• Any change in the nucleotide sequence of DNA
• Types of Mutations
• Mutations may Result from:
– Random errors in DNA replication
– Viruses
– Chemicals/toxins (cigarette smoke)
– Radiation (e.g. U.V. light, X-rays)
DF508 deletion: the most common cause of cystic fibrosis
Mutation responsible for Sickle Cell Anemia:
nucleotide substitution
Glu
Val
3 Types of Mutations:
Base Substitutions, Insertions or Deletions
• Base substitutions
– May result in changes in
the amino acid sequence
in a protein, or
– May be silent (have no
effect) – Why?
Substitute C for U
Met
Lys
Phe
Gly
Ala
AACGGUUAUCCU
Asn – Gly – Tyr – Pro
Met
Phe
Ser
Ala
Types of Mutations: Base Insertions and deletions
• Changes the
reading frame of
the genetic
message
mRNA
Protein
Met
Lys
Phe
Gly
Ala
(b) Nucleotide deletion
Met
Lys
Leu
Ala
His
• Although mutations
are often harmful
– They are the source
of the rich diversity
of genes in the
living world
– They contribute to
the process of
evolution by
natural selection