Transcription and Translation Laboratory Exercise
Introduction
Central Dogma
The central dogma of biology is below.
Protein synthesis requires two steps: transcription and translation. These steps are indicated in red on the diagram above. DNA structure and DNA replication were studied in the previous exercise. Be sure that you understand DNA structure and replication before continuing with this exercise.
DNA contains codes
Three bases in DNA code for one amino acid. The DNA code is copied to produce mRNA. The order of amino acids in the polypeptide is determined by the sequence of 3-letter codes in mRNA.
DNA vs RNA
Differences between DNA and RNA are listed in the table below.
DNA
RNA
Sugar:
deoxyribose
ribose
Bonds with Adenine:
thymine
uracil
# of Strands:
two
one
Kinds of RNA
Messenger RNA (mRNA)
Messenger RNA contains genetic information. It is a copy of a portion of the DNA.
It carries genetic information from the gene (DNA) out of the nucleus, into the cytoplasm of the cell where it is translated to produce protein.
Ribosomal RNA (rRNA)
This type of RNA is a structural component of the ribosomes. It does not contain a genetic message.
Transfer RNA (tRNA)
Transfer RNA functions to transport amino acids to the ribosomes during protein synthesis.
DNA Workshop Activity
Do the DNA Workshop Activity at http://www.pbs.org/wgbh/aso/tryit/dna/index.html#
Transcription
During transcription, mRNA is synthesized following the sequence of nucleotides in the coding strand of DNA. This will require the enzyme RNA polymerase and nucleotides from which RNA will be constructed. RNA polymerase will unwind the DNA, separate the strands, and assemble the mRNA molecule using the coding strand of DNA as a template.
Plastic beads will be used to represent the components of RNA indicated in the table below.
Model
ComponentChemical Represented # Needed Pink Ribose sugar 24 Red Phosphate group 24 Purple Uracil (U) 6 Orange Adenine (A) 6 Green Guanine (G) 6 Blue Cytosine (C) 6 RNA polymerase model sheet RNA polymerase 1 DNA model sheet DNA 1 These beads will be assembled to form 24 ribonucleotides. Recall that a nucleotide contains a base, a sugar, and a phosphate group.
Obtain the beads indicated in the table above. In addition, you will need a DNA molecule printed on a plastic strip and a plastic sheet representing an RNA polymerase molecule.
Assemble 6 ribonucleotides containing ribose, a phosphate group, and uracil as shown in the photograph below. Do not link the nucleotides together in a chain.
Assemble 6 nucleotides containing adenine. Again, do not link the nucleotides together.
Assemble 6 nucleotides containing guanine.
Assemble 6 nucleotides containing cytosine.
The plastic strip representing DNA has two holes punched into each end. A cut should be made between the holes so that the two strands of DNA can be separated. If the strip has not been cut, use scissors to make the cut.
Tape the plastic strip representing the DNA model to the top of the table.
RNA polymerase recognizes a specific base sequence in the DNA called a promoter and binds to it. The promoter identifies the start of a gene, which strand is to be copied, and the direction that it is to be copied.
The RNA polymerase model should be cut out from a plastic sheet. If it has not been cut out, use scissors to cut it out.
Slide the RNA polymerase model between the strands of the DNA model as shown in the diagram below.
RNA polymerase moves toward the 5' direction of the antisense (template) strand of DNA. As it moves, it will separate the two strands of DNA. The DNA strands will become reattached immediately behind the RNA polymerase as it passes by.
The first DNA nucleotide on the template strand after the promoter region contains a "T". Find the RNA nucleotide that is complimentary to this "T" and place it in the active site of the RNA polymerase model so that it "binds" to the "T" of the DNA. The nucleotide should be antiparallel to the DNA strand, that is, the 5' end should be on the left.
Slide the RNA polymerase molecule down one nucleotide but do not move the DNA and the RNA nucleotide that is currently in the active site.
Find the RNA nucleotide that is complimentary to the next DNA nucleotide and place it on the active site of the RNA polymerase. Remember to keep the phosphate groups toward the left.
Connect the phosphate group of this nucleotide to the ribose of the previously added nucleotide.
Slide the RNA polymerase down one nucleotide and add the third RNA nucleotide.
Bond (connect) this nucleotide to the two that are already there.
Repeat this procedure one nucleotide at a time until you reach the termination region of the template strand.
As the mRNA strand grows, it detaches from the DNA template.
Base sequences in the termination region signal the RNA polymerase to detach from the DNA and the mRNA is released. When you reach the termination region, remove the RNA polymerase and mRNA strand from the DNA model. Your mRNA should have 24 nucleotides.
The mRNA molecule that you constructed will be used in the next exercise.
Concepts
Be sure that you can answer the following questions before you begin the next section.
What did you simulate in this activity?
What is the name of this process?
In what part of the cell does this process occur?
What is the overall goal of the processes being studied today?
If the coding strand of DNA has the sequence listed below, what will be the sequence of nucleotides in the mRNA produced from that strand?
A A T G A C T C G
Translation
The information stored in the mRNA molecule that you constructed in the previous exercise will be used in this exercise to construct protein. This process is called translation.
This exercise will require the following materials.
Model
ComponentStructure Represented # Needed White oval beads Transfer RNA (tRNA) 15 White twist beads Amino acid 15 Ribosome model Sheet Ribosome 1 Arrange the mRNA molecule on your table so that the 5' end is on the left and the 3' end is on the right. The bases should be oriented so that they extend away from you.
The ribosome model should be cut out from a plastic sheet. If it is not already cut out, use scissors to cut it out.
Although there are 20 different amino acids and 64 different types of transfer RNA we will use only seven. If the beads representing amino acids (white twist beads) are not labeled, label seven of them. Each should have one of the following labels: fMet, Val, Tyr, Leu, Thr, Pro, and Asp.
If the beads representing transfer RNA (white oval beads) are not labeled, label seven of them. Each should have a "tRNA" label. In addition, each should have one of the following labels: fMet, Val, Tyr, Leu, Thr, Pro, and Asp.
Attach each amino acid to the appropriate transfer RNA molecule by snapping them together.
You should have seven amino acid-tRNA complexes similar to the one shown above.
Position the ribosome on (underneath) the 5' end of the mRNA strand as shown in the photograph below.
The first codon of the mRNA strand should be on the P site of the ribosome. The second codon should be on the A site.
The first codon is AUG. The second codon is GUC. Use the table below to find which tRNA and amino acid corresponds to these two codons.
First
Base
Second Base
Third
Base
U
C
A
G
U
UUU
phenylalanine
UCU
serine
UAU
tyrosine
UGU
cysteine
U
UUC
phenylalanine
UCC
serine
UAC
tyrosine
UGC
cysteine
C
UUA
leucine
UCA
serine
UAA
stop
UGA
stop
A
UUG
leucine
UCG
serine
UAG
stop
UGG
tryptophan
G
C
CUU
leucine
CCU
proline
CAU
histidine
CGU
arginine
U
CUC
leucine
CCC
proline
CAC
histidine
CGC
arginine
C
CUA
leucine
CCA
proline
CAA
glutamine
CGA
arginine
A
CUG
leucine
CCG
proline
CAG
glutamine
CGG
arginine
G
A
AUU
isoleucine
ACU
threonine
AAU
asparagine
AGU
serine
U
AUC
isoleucine
ACC
threonine
AAC
asparagine
AGC
serine
C
AUA
isoleucine
ACA
threonine
AAA
lysine
AGA
arginine
A
AUG (start)
methionine
ACG
threonine
AAG
lysine
AGG
arginine
G
G
GUU
valine
GCU
alanine
GAU
aspartate
GGU
glycine
U
GUC
valine
GCC
alanine
GAC
aspartate
GGC
glycine
C
GUA
valine
GCA
alanine
GAA
glutamate
GGA
glycine
A
GUG
valine
GCG
alanine
GAG
glutamate
GGG
glycine
G
The first codon of mRNA in all organisms is AUG. According to the table above, this codon (AUG) codes for methionine.
Place the fMet tRNA complex on the P site of the ribosome. This tRNA has three nucleotides that are complimentary to the AUG and binds to AUG. Our model bead does not show these three nucleotides or anticodon.
Attach the tRNA to the "U" of the mRNA. Keep in mind that in reality, it is also attached to the "A" and the "G".
Determine which amino acid corresponds to the codon that is in the A site of the ribosome.
Attach the appropriate tRNA and its amino acid to the mRNA in that site.
A bond now forms between the amino acid in the P site and the one in the A site. Remove the fMet from the tRNA in the P site and attach it to the amino acid in the A site as illustrated below.
The tRNA that remains in the P site moves away from the site and can be reused by attaching to another fMet.
Remove the tRNA from the P site and slide the ribosome three bases (one codon) to the right.
Read the codon and attach the correct tRNA-amino acid.
Remove the two amino acids in the P site and attach them to the amino acid in the A site.
Remove the tRNA that remains in the P site and slide the ribosome three nucleotides to the right.
Repeat this procedure until a stop (termination) codon is reached. When a stop codon is reached, the protein chain is released and the ribosome becomes detached from the mRNA molecule.
Before leaving the lab, disassemble the mRNA into nucleotides and disassemble the polypeptide chain.
Concepts
State the purpose (the goal) of the procedure that you just completed above.
What is the name of the procedure?
In what part of the cell does this procedure occur?
Each column in the table below represents three nucleotides. In each column, fill in the information in the cells that are blank by using the information in the cell that is given.
Template strand GGG
Non-coding strand TAC
mRNA CCU
tRNA UCG
Amino Acid Leu
