Download Protein Synthesis Lab: Day #1

HASPI Medical Biology Lab 02
Chromosomes are simply portions of DNA wound up and
organized into a form that makes it easier for cells to find
the directions, or gene, that it needs to make a specific
protein. Different organisms have a different number of
chromosomes depending on the amount of DNA, or
instructions, needed to build and keep that organism
functioning. Humans normally have two sets of 23
chromosomes. One set comes from each parent with the
same genes, but with different versions of those genes.
If they are the same, why do we have two sets? Although each chromosome has the same genes
that contain the directions for the corresponding protein, these genes can vary slightly and create
the differences we see among humans. For example, the gene for eye color that a child may inherit
could be blue, brown, green, or hazel. You will learn more about chromosome structure and
inheritance in a later activity.
From DNA to Protein
Now that you understand that DNA contains the
code for proteins, the question becomes how the
code in DNA actually leads to proteins? This process
is incredibly complex, but can be summarized in three
steps: transcription, protein synthesis or translation,
and protein folding.
Transcription
DNA is very fragile and it is vital that not be damaged.
For this reason, our bodies have created a way to
make a copy of DNA, specifically a gene, so that it
doesn’t have to leave the protection of the nucleus.
The copy is made out of RNA, or ribonucleic acid,
called messenger RNA, or mRNA. This copying
process is called transcription and ONLY occurs in the
nucleus.
Protein Synthesis or Translation
Once an mRNA copy of the gene has been created,
the ribosome can build a protein using the mRNA
copy as directions. The ribosome translates the order
of amino acids in the protein and bonds them
together into a chain.
Protein Folding
The length of the amino acid chain produced by ribosomes can range from only a few hundred to
hundreds of thousands of amino acids long. The amino acid chain is transported
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to the endoplasmic reticulum (ER) where they are folded and can even have carbohydrates or lipids
added to them to produce functioning proteins. An amino acid chain cannot perform a function
until it has been folded into its functional shape.
Amino acid chains are also known as polypeptide chains. The
interactions and bonds that occur between the different amino acids
are what cause the folding and shaping of the protein. Every amino
acid has a functional side that can cause or prohibit bonding with
other amino acids. Proteins, called chaperones, can assist an amino
acid chain during the folding and bonding process to create a
finished protein that can now perform a function in the body.
Cystic Fibrosis
Cystic fibrosis is a common genetic disease
caused by a mutation in a gene called the
cystic fibrosis transmembrane conductance
regulator (CFTR) gene. The CFTR gene is located
on chromosome 7 and has the directions to
create the CFTR protein. The CFTR protein is a
channel protein that regulates how salts, most
commonly sodium (Na+) and chloride (Cl-), and
water move through the cell membranes of
epithelial cells. Epithelial cells cover surfaces of
the body and can be found in the skin,
respiratory, and digestive tracts.
Na+ and Cl- help control the movement of
water into tissues. When the CFTR protein does
not function correctly, chloride (Cl-) is unable to pass through the center channel and sodium (Na+)
is also unable to pass through the cell membrane. When they are imbalanced, watery substances
like mucus are unable to move into the tissues and the mucus becomes extremely sticky and thick.
(The role of mucus is to lubricate the surfaces of the body.) As a result, symptoms of cystic fibrosis
include:
 Extremely salty skin
 Thick, sticky mucus that can block respiratory and digestive tracts
 Frequent respiratory infections due to bacteria trapped in mucus
 Wheezing, persistent cough, and shortness of breath
 Lack of digestion leading to poor growth/weight
The cystic fibrosis mutation is a recessive disorder passed from parent to offspring. This means an
individual needs two copies of the mutated CFTR gene to have cystic fibrosis. Since the
mutation is recessive, a parent may not have symptoms of cystic fibrosis or know they carry the
mutation. There are more than 30,000 people in the U.S. with cystic fibrosis and more than 1,000
cases are diagnosed yearly. More than 10 million people in the U.S. are carriers of cystic fibrosis.
When cystic fibrosis was first discovered, few sufferers lived past 6 years old, but due to medical
advances the median age of survival has increased to 37 years old.
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Genes, Proteins, and Disease; HASPI Medical Biology Lab 02
Review Questions
1. What are proteins? Give 3 examples of functions they perform. (see “Macromolecule” notes)
2. How are DNA, genes, chromosomes, and proteins related?
3. Describe protein folding. What causes an amino acid chain to fold? (see “More on Protein”
notes)
4. What is a mutation? Explain how a mutation in a gene can influence the protein it creates.
5. List and explain 2 types of mutations.
6. What is cystic fibrosis? List 3 symptoms associated with cystic fibrosis.
7. What is the purpose of the CFTR protein?
8. What happens when the CFTR protein is mutated?
9. How does an individual get cystic fibrosis?
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Purpose
Create a model to simulate the process by which a protein is produced, and how a mutation can
impact a protein’s function.
Background
DNA contains the directions to create the proteins that allow
our bodies to function. Portions of the DNA that contain
directions for a single protein are called genes. Because
DNA is delicate we DO NOT want to remove it from the
nucleus and instead it makes a copy of the directions using
RNA. RNA polymerase is a protein that copies the DNA and
the finished copy is made of messenger RNA, or mRNA. This
copying process is called transcription. After transcription,
the copy leaves the nucleus and is in the nucleus with
special organelles called ribosomes. The ribosomes translate
the directions from the copy to build a protein in a process
called translation. Lastly, the protein must fold into its final
shape in order to be able to perform a function.
In this activity, your team will be making a copy of
the CFTR gene that contains the directions for
creating the CFTR protein. This protein is a transport
protein that embeds itself in the cell membrane
and regulates certain substances (Na+ and Cl-)
moving in and out of the cells in the skin, pancreas,
and lungs. If this protein is not built correctly, and
therefore not able to function correctly, these
substances cannot move in/out of the cell and
cause a thickening and build-up of mucus. This
causes the condition known as cystic fibrosis.
Procedure/Directions
Your lab team will be given tasks, or directions, to perform on the left. Record your questions,
observations, or required response on the right when space is available.
Part A: Set-Up
Task
Response
Obtain the following supplies:
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4
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Scissors
Tape
Normal CFTR Gene sheets (2 pgs)
Mutated CFTR Gene sheets (2 pgs)
Blank white paper for mRNA (transcription)
2 RNA Polymerase sheets (1 pg)
2 Ribosome sheets (1 pg)
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Cut out the “Normal CFTR Gene” and “Mutated CFTR
Gene” strips along the dotted line and tape each end
together. Make sure to tape the correct ends to one
another to create a single DNA strand of the normal
CFTR gene AND the mutated CFTR gene (see image.)
You will transcribing the mRNA on the blank white
sheets of paper from your supplies you’ve obtained.
Turn the paper landscape and cut this paper into one
inch strips. You will be taping these strips together
using the amount of strips it takes to transcribe the
DNA.
The “tRNA templates” have been cut out for you and
are in the bowls of beads that correspond to the
correct amino acids. (The beads are the amino acids)
The RNA Polymerase and Ribosome templates have
been cut out for you.
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Part B: Transcription
Task
1
Get into a team of 4. Within your team separate
into pairs. One pair will be following the procedure
with the “Normal CFTR Gene” and the other pair will
be following the EXACT same procedure with the
“Mutated CFTR Gene.”
2
In this part of the activity, your team will be
transcribing the normal and mutated CFTR gene
that contains the directions for creating the CFTR
protein.
Response
Mutated
Genes, Proteins, and Disease; HASPI Medical Biology Lab 02
Normal
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a. Where does transcription occur?
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RNA polymerase is the protein that functions to
unzip, or open, the DNA double helix and bond RNA
nucleotides as they match up to the DNA
nucleotides creating the mRNA strand. Remember
Cytosine bonds to Guanine and Adenine bonds to
Thymine. In RNA, Thymine becomes Uracil.
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a. What are the 4 DNA nucleotides?
b. What are the 4 RNA nucleotides?
c. Which nucleotides complementary base pair
with one another in DNA? In RNA?
1. DNA:
2. RNA:
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Place your “RNA Polymerase” template on the table
(each pair of team members need a template).
Feed the START end of your CFTR gene into and
through the cut in the RNA polymerase sheet (see
image).
Starting at the START end of the CFTR gene, place the blank white paper strips next to the CFTR
genes. On the white paper strips, write the correct RNA nucleotides that base pair with the DNA
nucleotides. You are creating your mRNA strand! For example, the Adenine DNA nucleotide will
match with a Uracil RNA nucleotide.
As you move down the CFTR gene, slide it through the RNA polymerase. Continue this process of
copying the DNA nucleotides matching RNA nucleotides, as you write them on the white paper
strips (mRNA strand), until you reach the end of the CFTR gene. Tape your white paper strips
together as you run out of room completing each strip. Make sure to tape them in order!
Remove the CFTR gene from the RNA polymerase. You have completed an RNA copy of the
CFTR gene. This copy is called messenger RNA, or mRNA. In an actual cell, this process would be
repeated with the same CFTR gene and RNA polymerase many times, to create multiple mRNA
copies.
Genes, Proteins, and Disease; HASPI Medical Biology Lab 02
a. What is the function of RNA polymerase?
b. What is mRNA? Why is it important to create mRNA rather than use actual DNA for the next step?
c. Summarize the transcription process.
Part C: Translation
Task
1
2
3
Response
In this part of the activity, your team will be using the
mRNA copy of the CFTR gene you created in
transcription to decode the order of amino acids that
make up the CFTR protein.
a. What is the purpose of translation?
To simulate the structure of proteins, you will be using plastic beads to represent the 20 amino
acids that make up protein. The plastic beads (amino acids) are in the glass jars on the back
counter.
Before you build your protein by collecting the amino
tRNA
mRNA
acids, place your “Ribosome” on the table. Feed your
mRNA copy into the ribosome with the written
nucleotides facing up.
Only the first three mRNA nucleotides should be visible in
the window of the ribosome.
rRNA
Note: Your mRNA is hand written on the
white paper strips and does not look the
the mRNA in this picture; the tRNA’s in this picture are in
the bowls of plastic beads (amino acids)
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!
!
!
!
!
!
Phenylalanine
Phenylalanine
Leucine
!
!
!
!
!
!
tRNA
tRNA
tRNA
!
!
!
Every three mRNA nucleotide bases are called a codon.
Each
codon
is
actually
a
3-base
code
A
A
! A
! A
A
A
A
G !
for a specific amino acid. There are 61 possible codons
means
! that code for 20
! amino acids. This
!
!
some amino acids have more than one codon.
!
!
!
!
!
!
!
!
!
!
!
!
Using your mRNA codon chart, write the correct amino! acid under each
codon
on
your
!
! mRNA
!
!
!
strand (white paper strip).
Leucine
Leucine
Leucine
!
!
!
!
tRNA
!
tRNA
!
tRNA
!
!
!
Obtain two pipe cleaners
A
A
A
G ! G
A
! G
! G
!
!
!
!
Transfer RNA (tRNA) float around in the cytoplasm near!
!
!
!
!
ribosomes and the endoplasmic reticulum. One end of!
!
!
!
!
!
!
a tRNA molecule has an amino acid attached and the!
!
!
!
!
!
other end has a set of 3 bases that can match up to an
Isoleucine
Isoleucine
Isoleucine
!
!
!
mRNA codon. This 3-base code located on tRNA is
!
!
!
tRNA
tRNA
tRNA
!
!
!
called the anticodon.
A
A
A
G ! U
A
! U
! U
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
Using your amino acid sequence you wrote on your mRNA
strand below
the
!
! the codons, locate
!
!
!
corresponding beads and string them onto your pipe cleaner
in the correct
order.
See !step 9 Valine
Valine
Valine
!
!
!
below before you start!
!
!
!
tRNA
tRNA
tRNA
!
!
!
A
A
A
G ! C
A
! C
! C
For example, the first mRNA codon is “AUG” so the anticodon
is “UAC”,! which codes for! the
!
!
methionine amino acid.
C
U
amino
acid
7
U
anticodon
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Slightly bend the end of the pipe cleaner to prevent the
amino acids/beads from sliding off the end. Place the
amino acid/bead on your pipe cleaner and slide it to
the end (see image).
Continue this process until you reach the end of the
10 mRNA copy and hit a STOP codon. Fold the end of the
pipe cleaner to prevent the amino acids/beads from
falling off. You have just created an amino acid chain.
a. Summarize the translation process.
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Genes, Proteins, and Disease; HASPI Medical Biology Lab 02
U
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
Part D: Protein Folding
Task
1
Response
So far you have made an mRNA copy of the CFTR gene
and decoded the copy to determine the amino acid
order. You have an amino acid chain, but it is not yet a
functional protein. Different amino acids interact and
bond with each other causing the amino acid strand to
fold and create a protein that can perform a function.
http://www.piercenet.com/media/ProStructureFig1.gif
glue dot
2
Obtain a glue dot sheet (share among your team).
Place a glue dot in each of the 6 spaces of the
methionine amino acid/bead (see image).
glue dot
glue dot
glue dot
glue dot
glue dot
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4
For your amino acid strand, methionine and glycine will
bond to each other. (both are clear in color)
Starting at methionine, follow the amino acid chain until
you find a clear bead (glycine). Fold the amino acid
strand and connect glycine to a space in methionine at
one of the glue dots (see image).
methionine
glycine
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Continue down the amino acid chain, folding and
connecting any glycine to the next open space on
methionine, until all of the glycine amino acids on the
chain have been attached.
Mutated
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You now have a completed protein!
Normal
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Part E: Protein Function
Task
1
Now that your team has completed a normal and
mutated CFTR proteins, compare their structures.
2
CFTR functions to move salts in/out of cells. You
have only created a portion of this protein that
attaches and anchors the remaining protein to the
cell membrane. The complete CFTR protein is
actually more than 1,300 amino acids long!
3
The part of the normal CFTR protein that you
created is responsible for connecting the CFTR
protein to the cell membrane. The green portion of
your normal protein would be the active site that
binds to a portion of the cell membrane.
4
Your instructor has taped up a simulated cell
membrane. Take your normal and mutated CFTR
proteins to the simulated cell membrane and see if
the green active site binds and stays attached to
the green attachment point on the cell membrane
(see figure to the right)
Response
a. Compare and contrast the structure of your
normal and mutated CFTR proteins.
Med Bio Lab 02: The Cell Membrane
The cystic fibrosis transmembrane conductance regulator (CFTR) protein creates a channel for chloride ions (Cl -) to move
through the cell membrane of epithelial cells lining the lungs, pancreas, skin, and other surfaces of the body.
·
NORMAL PROTEIN: If your CFTR protein is normal and attaches the cell membrane, it will allow Cl – to move through
the cell membrane and maintain a balance of ions on the outside and inside of the cell. As a result of this balance,
mucus within the airways hydrated and functioning correctly.
·
MUTATED PROTEIN: If your CFTR protein is mutated and DOES NOT attach or open Cl – cannot move through the cell
membrane. This creates an imbalance in the amount of ions on the outside and inside of the cell membrane and
affects the amount of water in the cell. As a result, mucus becomes extra sticky and is difficult to remove from the
lungs. This includes any bacteria trapped in the mucus, which can cause numerous respiratory infections. This is only
ONE example of a symptom of cystic fibrosis.
!
!
!
!
Cl#$#
!
Outside Lung
Airway
!
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You have completed this activity! DNA RNA 
protein is known as the central dogma of genetics,
and you now have first-hand experience as to how
this process happens. In actuality, your cells can
transcribe a gene that is 1,500 base pairs and
produce the protein it codes for within 1.2 seconds!
Cl#$#
Cl#$#
Cl#$#
Cl#$#
Cell Membrane
Inside Lung Airway
mucus!
mucus!
Genes, Proteins, and Disease; HASPI Medical Biology Lab 02
mucus!
Analysis & Interpretation
Analysis Questions
1. Compare and contrast how the normal and mutated proteins adhered to the cell membrane?
How did the mutation impact the function of the CFTR protein?
2. What happened to the green active site (place where the reaction takes place) of the protein
in the mutated CFTR protein? Why is this a problem?
3. Hypothesize what might happen if the deletion mutation was at the end of the gene rather
than towards the beginning.
4. Explain how the CFTR gene leads to the creation of the CFTR protein?
5. Construct an explanation based on evidence for how the structure of DNA determines the
structure of proteins, which carry out essential functions of life through systems of specialized
cells.
Genes, Proteins, and Disease; HASPI Medical Biology Lab 02
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