Biology 1334 Lab Assignment #2

DNA and Protein Synthesis Hands-On Labs, Inc. Version 42-0051-00-02

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 (Guanine- Cytosine).

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 anti- codons 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.

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Experiment DNA and Protein Synthesis