structure and function of DNA. DNA and Protein Synthesis

structure and function of DNA.DNA and Protein Synthesis
Review the safety materials and wear goggles when
working with chemicals. Read the entire exercise
before you begin. Take time to organize the materials
you will need and set aside a safe work space in
which to complete the exercise.
Experiment Summary:
You will learn the structure and function of DNA and
RNA. You will learn the similarities and differences
between DNA and RNA. You will learn the process
of protein synthesis and create and use models to
demonstrate both transcription and translation.
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EXPERIMENT
Learning Objectives
Upon completion of this laboratory, you will be able to:
● Review the structure and function of DNA.
● Identify the codons that code for amino acids in DNA and RNA.
● Explain the purpose of start and stop codons in protein synthesis.
● Summarize the steps involved in protein synthesis and define a ribosome and its three sites.
● Summarize the steps of transcription, including: initiation, elongation, and termination.
● Summarize the steps of translation, including; initiation, elongation, and termination.
● Illustrate and model the processes of transcription and translation.
● Construct a series of tRNA molecules and write the anti-codons and amino acids each tRNA
carries.
● Explain the difference in the number of amino acids that were present at the start and at the
end of the translation model.
Time Allocation: 3 hours
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Experiment DNA and Protein Synthesis
Materials
Student Supplied Materials
Quantity Item Description
1 Camera, digital or Smartphone
1 Pair of scissors
1 Printer
10 Sheets of printer paper
1 Pen or pencil
1 Tape
HOL Supplied Materials
Quantity Item Description
1 DNA Nucleotide Template
1 RNA Nucleotide Template
1 tRNA Template
Note: To fully and accurately complete all lab exercises, you will need access to:
1. A computer to upload digital camera images.
2. Basic photo editing software such as Microsoft Word® or PowerPoint®, to add labels, leader
lines, or text to digital photos.
3. Subject-specific textbook or appropriate reference resources from lecture content or other
suggested resources.
Note: The packaging and/or materials in this LabPaq kit may differ slightly from that which is listed
above. For an exact listing of materials, refer to the Contents List included in your LabPaq kit.
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Experiment DNA and Protein Synthesis
Background
DNA, Codons, and Proteins
Deoxyribonucleic acid (DNA), the genetic material of all living organisms, is composed of two
chains of nucleotides wound together in a double-helical formation. Nucleotides, the molecules
responsible for the structural units of DNA, are composed of three sections: a phosphate
group (PO4
), a sugar (deoxyribose) group, and a nitrogenous base. There are four different DNA
nucleotides: adenine (A), thymine (T), cytosine (C), and guanine (G), which are identical in their
phosphate and sugar groups, but vary in their nitrogenous bases. The bonds between the sugar
and phosphate groups of each nucleotide form the sugar-phosphate backbone of DNA and the
two strands wind together as a result of base pairing: AT (Adenine-Thymine) or GC (GuanineCytosine).
The arrangement of the four DNA nucleotides creates the genetic code, the blueprint for all living
things. The genetic code is composed of codons, triplets of nucleotides that contain the code for
the production of amino acids, which are strung together to create proteins (polypeptide chains).
Proteins are highly complex, organic substances that provide a vast number of functions in living
organisms, including maintenance of cells and growth. Thus, proteins are essential components
of living tissues including: skin, bones, and muscle. The four different nucleotides provide 64
different codons (four options for each of three positions = 43
= 64 options), which code for one
of twenty amino acids or a stop codon. See Table 1.
Table 1. Codon Chart (DNA)
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Experiment DNA and Protein Synthesis
Protein Biosynthesis and Transcription
Protein biosynthesis is the process where cells use the genetic code to build proteins. The process
differs slightly between prokaryotes (single-celled organism with no organelles or distinct nucleus)
and eukaryotes (single or multi-celled organisms with organelles and DNA contained in a distinct
nucleus). In the context of this experiment, the focus will be on the main steps and commonalities
between the prokaryotic and eukaryotic protein biosynthesis steps. There are two main steps in
protein biosynthesis: transcription and translation.
Transcription is the process by which single stranded RNA is synthesized from DNA. RNA
(ribonucleic acid), like DNA, is composed of nucleotides with a phosphate, a sugar (ribose), and
one of four nitrogenous bases. The nucleotides adenine, cytosine, and guanine exist in both DNA
and RNA, however; in RNA, the nucleotide uracil (U) replaces thymine (T), and binds with adenine
(A). See Figure 1.
Figure 1. Nitrogenous bases in DNA and RNA. Note that while DNA is double-stranded, RNA
exists as a single-strand. © udaix
There are three steps in transcription: initiation, elongation, and termination. A general depiction
of transcription is shown in Figure 2.
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Experiment DNA and Protein Synthesis
Figure 2. General schematic of transcription. © The National Human Genome Research Institute
In initiation, an enzyme called RNA polymerase binds to the DNA promoter, which is the DNA
sequence that initiates transcription. The RNA polymerase causes the two strands of DNA to begin
unwinding and separate from one another. In elongation, the RNA polymerase travels downstream
(3’ to 5’) along the DNA antisense strand, elongating the mRNA transcript in the 5’ to 3’ direction.
The DNA antisense strand is the template strand from which the mRNA is transcribed. Figure
2 illustrates how transcription creates an mRNA copy of the DNA sense, or coding, strand, with
uracil replacing thymine in the newly constructed mRNA. As the RNA polymerase continues to
move downstream, the two strand of DNA re-wind into a double-helix formation. In termination,
the RNA polymerase detaches from the DNA and releases the transcribed mRNA. In a prokaryote,
the released mRNA is complete and ready to move into translation, while in a eukaryote the
released RNA undergoes a series of steps where it is processed before moving into translation as
mRNA.
Translation
Translation, the second main step of protein synthesis, is the process by which the mRNA (created
in transcription) is converted into a protein. In a prokaryote, translation occurs in the cytoplasm,
the same site as transcription; while in eukaryotes, translation occurs in the cytoplasm, where
it is carried after transcription has completed in the nucleus. There are two major players in
translation; transfer RNA (tRNA) and ribosomes. tRNA is a clover-shaped molecule that acts as the
interpreter between mRNA and the protein it will help to synthesize. A ribosome is an organelle
which functions as the site of protein synthesis. Ribosomes are made of ribosomal RNA (rRNA)
and protein molecules, and are divided into two subunits: large and small. The small ribosomal
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Experiment DNA and Protein Synthesis
subunit binds the mRNA and reads the information contained in the mRNA nucleotide sequence.
The large ribosomal subunit contains three binding sites: the peptidyl-tRNA site (P site), the
aminoacyl-tRNA site (A site), and the exit site (E site). See Figure 3.
Figure 3. The ribosome.
There are three steps in translation: activation and initiation, elongation, and termination. A
general depiction of translation is shown in Figure 4.
Figure 4. General schematic of translation.
In the first step of translation, activation and initiation, the mRNA is threaded between the
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Experiment DNA and Protein Synthesis
small and large subunits of the ribosome. The ribosome signals the start of translation when it
encounters and binds to the first start codon (AUG, which codes for the amino acid methionine)
at the A site. The tRNA carrying the anticodon (UTC) and the methionine binds to the mRNA
codon at the ribosomal A site, creating the initiation complex, signaling translation to begin.
The tRNA bound mRNA then moves into the P site, which brings in the next tRNA carrying the
complementary anticodon and amino acid to the mRNA now in the A site.
The amino acid in the P site then forms a peptide bond with the amino acid in the A site,
releasing the amino acid from the tRNA in the P site and moving the empty tRNA into the E site.
Simultaneously, the tRNA in the A site, holding the two peptide-bonded amino acids, then moves
into the P site, signaling the next tRNA to bind to the mRNA in the A site. Using Figure 4 as an
example, as the empty tRNA (which had been carrying valine) exits the E site, a peptide bond is
formed between lysine (in the P site) and cysteine (in the A site). The lysine (peptide bound to
valine, glutamate, serine, and glycine) then detaches from the tRNA in the P site and attaches to
the tRNA in the A site. The tRNA in the A site (now carrying the cysteine, lysine, valine, glutamate,
serine, and glycine) then moves into the P site, releasing the tRNA that had carried the lysine from
the E site. As the tRNA is released from the E site, tRNA carrying the anticodon AUA and the amino
acid tyrosine (yellow Tyr) binds to the mRNA in the A site, continuing the process. This continuous
process, called elongation, builds the polypeptide chain (protein) one amino acid at a time until
the mRNA reads a stop codon (UAA, UAG, or UGA).
When a stop codon is encountered in the mRNA at the A site, termination is signaled. In
termination the stop codon signals the end of elongation, which cleaves the protein from the
tRNA, allowing it to exit the ribosome. The two ribosome subunits and the mRNA then dissociate
from one another, completing the translation process. The protein then undergoes a series of
steps including post-translational modifications and protein folding to assume its new shape.
In 2009, Dr. Ada Yonath won the
Nobel Prize in Chemistry. She was the first
woman to win the Nobel Prize in Chemistry
in 45 years, since Dorothy Crowfoot Hodgkin
in 1964, and the first woman in the Middle East
to ever win the Chemistry Nobel Prize. Her award,
shared with Dr. Thomas Steitz and Dr. Venkatraman
Ramakrishnam, was the result of her work on the
structural determination of the ribosome, determining
the structure of both the small and large ribosomal
subunits. Her work lead to the conclusion that a
ribosome is a ribozyme (ribonucleic acid enzyme), that
organizes its substrates in the stereochemistry necessary
for the formation of peptide bonds. From her work
came the new and exciting crystallization technique
called cryo bio-crystallography, which allows for the
crystallization of large biological macromolecules at
cryogenic temperatures (approximately -320°F)
allowing the macromolecules to maintain
their solution state.
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Experiment DNA and Protein Synthesis
Exercise 1: Protein Synthesis
In this exercise, you will model the steps of protein synthesis, starting with a single strand of
nucleotides and ending with a protein.
1. Print 6 copies of the DNA Nucleotide Template, 4 copies of the RNA Nucleotide Template, and
1 copy of the tRNA Template. It is preferable, but not necessary, to print them in color. The
templates are located in the “Supplemental Documents” folder of your digital courseware.
2. Review the coding strand of DNA (5’ to 3’) in Data Table 1 of your Lab Report Assistant.
3. Create the template strand of DNA (3’ to 5’) and record in Data Table 1.
4. Gather the scissors, tape, and the 6 printed copies of the DNA Nucleotide Template. Cut out
the nucleotides from the template. It is not necessary to cut out the entire nucleotide; rather,
cut the nucleotide in a rectangular shape, only cutting out the details of the nitrogenous
bases. See Figure 5.
Figure 5. Cutting out DNA nitrogenous bases.
5. Using the DNA nucleotides, create the entire double strand of DNA by matching up and taping
together the base pairs. See Figure 6 as an example.
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Experiment DNA and Protein Synthesis
Figure 6. Pairing of DNA nucleotides.
6. Take a photograph of the completed double strand of DNA with your name and the data
showing in the photograph. Resize and insert the photograph into Data Table 2 of your Lab
Report Assistant. Refer to the appendix entitled “Resizing an Image” for guidance with resizing
an image.
7. Determine the mRNA strand that transcription would produce from the DNA template strand
and record the mRNA strand in Data Table 1.
8. Gather the 4 printed copies of the RNA Nucleotide Template. Cut out the nucleotides from
the template. It is not necessary to cut out the entire nucleotide; rather, cut the nucleotide in
a rectangular shape, only cutting out the details of the nitrogenous bases.
9. Using the RNA nucleotides, create the mRNA strand by matching up and taping together the
base pairs.
10. Take a photograph of the mRNA strand with your name and the date showing in the photograph.
Resize and insert the photograph in Data Table 2.
11. Starting with the first mRNA nucleotide, determine what amino acids the codons in the mRNA
are coding for and record in Data Table 1.
Note: Use Table 2 to determine the amino acids coded by RNA codons.
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Experiment DNA and Protein Synthesis
Table 2. Codon Chart (RNA)
12. Gather the printed copy of the tRNA Template and cut out the tRNAs.
13. Build the line of tRNAs that would flow into the A site during translation. Write the anticodons
into each tRNA and the amino acid the mRNA codes for. See Figure 7 as an example of
the tRNA that would be created from the mRNA codons CCU.
Note: Use Figure 4 in the Background section as needed to help organize your thoughts and identify
where in the mRNA strand translation would begin.
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Experiment DNA and Protein Synthesis
Figure 7. tRNA created from mRNA (CCU). Note that the anti-codons (GGA) and the name of the
amino acid (gly = glycine) are written into the tRNA.
14. Take a photograph of the tRNAs (in order) with your name and the date showing in the
photograph. Resize and insert the photograph in Data Table 2.
15. Write the name of the each amino acid in the final protein created from translation and record
in Data Table 1.
16. When you are finished uploading photos and data into your Lab Report Assistant, save your
file correctly and zip the file so you can send it to your instructor as a smaller file. Refer to the
appendix entitled “Saving Correctly” and the appendix entitled “Zipping Files” for guidance
with saving the Lab Report Assistant correctly and zipping the file.
Note: Use a textbook or internet source, as necessary, for a list of the three-letter amino acid
abbreviations and the full amino acid names.
Questions
A. How many amino acids were coded for by the mRNA? How many amino acids were present
in the final protein chain created in translation? In detail, explain the differences in the two
numbers; why were some amino acids coded for by the mRNA but not present in the final
protein chain? What amino acids were omitted from the final protein chain? Explain your
answers.

Experiment DNA and Protein Synthesis

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