Week 5 Lab: Gene Expression -

To delve a bit deeper into our topic this week, we are going to spend some time using Phet’s Gene Expression Simulation, as well as work through the effects of various mutations on gene expression. To access the simulation, you can either click on the Gene Expression Simulation Link below (recommended) or use the version that is embedded in each of the parts below.

HERE IS THE LINK https://phet.colorado.edu/sims/html/gene-expression-essentials/latest/gene-expression-essentials_en.html

Gene Expression Lab Simulation worksheet

Objectives:

● Identify the roles transcription factors, RNA polymerase, ribosomes, and mRNA destroyers have on transcription and translation.

● Distinguish between the location and function of regulatory regions compared to transcribed regions of DNA.

● Predict the effects of concentration, affinity, and degradation rates of transcription factors and RNA polymerase on gene expression.

● Identify the effects of mutations on gene expression. Background: Transcription is the process of making mRNA from DNA. This is a highly regulated process that our cells complete in preparation to make a protein. Translation is the process of making a protein from a piece of mRNA.

DNA ——————–> mRNA ——————–> protein transcription translation

Not all regions of DNA are used to make mRNA – only the parts of DNA that correspond to genes. Even then, not all gene regions are transcribed all the time. When genes are transcribed into mRNA depends on the needs of the cell. Once mRNA is made from DNA, it is translated into protein. Translation is an energy expensive process (it requires LOTS of ATP) which is one reason the cell only completes the process when the protein product is needed. This week’s “Reading and Lesson” explains many of the details of these highly complicated processes, transcription and translation. Please review the lesson for a deeper understanding of the concepts in this lab activity. Procedure: Click the Play arrow on this Gene Expression activity to complete the simulations. (The simulations are also embedded in the Canvas lab assignment page.) You will complete 3 simulations: 1) Expression, 2) mRNA, and 3) Multiple Cells.

Part 1: Expression Simulation

Click “Expression” to start that simulation. Notice the molecule that spans across the screen, from left to right. Answer the following 2 questions:

1. What is this molecule that spans across the page that is shown in red and blue?

2. What do you think the different colors (red and blue) of the molecule represent?

https://phet.colorado.edu/en/simulation/gene-expression-essentials
Now, start the process of transcription.

For transcription, you need these things to happen. First, most genes require 1 or 2 “transcription factors” to bind to the area in front of the gene (called the “regulatory region”). Second, an RNA polymerase (an enzyme that makes mRNA from DNA) needs to be present in order for transcription to occur.

1. Drag one Positive Transcription Factor and one RNA Polymerase from the box called Biomolecule Toolbox to the regulatory region on the DNA molecule. This should start TRANSCRIPTION.

2. Now, drag a ribosome next to the mRNA, in order to do TRANSLATION. 3. The mRNA is eventually broken down by an mRNA destroyer protein. Drag one of these next to the

mRNA when it is done making a protein. 4. Put the protein in Your Protein Collection. 5. Stop the gene from working by dragging the Negative Transcription Factor to the Regulatory Area, and

remove the Positive Transcription Factor by dragging it out of the way.

After you have made 1 protein, answer these 5 questions. HINT: Think about what/where things are at the start, and what/ where things are at the end of the process.

1. What does the “Positive Transcription Factor” do?

2. What does the “RNA Polymerase” do?

3. What does the “Ribosome” do?

4. What does the “mRNA destroyer” do?

5. What does the “negative transcription” factor do?

Click the yellow “Next Gene” box to begin working on the second gene. Can you remember the steps in order from your first trial? Try to see if you can! (HINT: There is one small difference between the transcription of gene 2 versus gene 1 – the difference is not in the order of steps but in the amount of something!) If not, not to worry, we are still learning… As a reminder, the steps are:

1. Drag Positive Transcription Factors and one RNA Polymerase from the box called Biomolecule Toolbox to the regulatory region on the DNA molecule. This should start TRANSCRIPTION!

2. Now, drag a ribosome next to the mRNA, in order to do TRANSLATION! 3. The mRNA is eventually broken down by an mRNA destroyer protein. Drag one of these next to the

mRNA when it is done making a protein. 4. Put the protein in Your Protein Collection. 5. Stop the gene from working by dragging the Negative Transcription Factor to the Regulatory Area, and

remove the Positive Transcription Factors by dragging them out of the way.

After you have made the second protein, answer these 2 questions.

1. What is one difference you noticed that was required to initiate the transcription of gene 2 versus gene 1?

2. What could be an advantage of multiple positive transcription factors versus only one?

Now, put all of your items back in the Biomolecule Toolbox and begin again, and answer the following 2 questions.

1. What happens if you add 2 RNA Polymerases (one after the first, before transcription is complete), and then 2 ribosomes (one for each mRNA)?

2. What would be the benefit of working this way versus adding RNA Polymerase one at a time?

Click the yellow “Next Gene” box to begin working on the third gene. Can you remember the steps in order from your first trial? Try to see if you can!

Additional 4 Questions from the Expression Simulation:

1. What is gene expression?

2. What molecules are involved in gene expression? List them all and state the role of each.

3. What is the difference between the “regulatory region” and the “transcribed region”?

4. A student says that “ALL DNA codes for proteins.” Do you agree with her? Why or why not? Give evidence to support your answer.

Part 2: mRNA Simulation

At the bottom of the simulation page, click on the next simulation (it’s greyed out) called mRNA.

You should see a strand of DNA with a bunch of RNA Polymerases floating around. (If the RNA Polymerases are not moving, click the Play button.) Answer the following 7 questions.

1. Is mRNA being made?

2. In the Positive Transcription Factor box, slide the Concentration slider from NONE to just a tad (a couple millimeters or so) away from NONE. What do you notice is happening in the simulation now?

3. Move the Concentration slider all the way to HIGH. How does this affect what is happening in the simulation?

4. Leave the Concentration slider on HIGH but move the Affinity slider all the way to LOW. What happens? Move the Affinity slider to a midway setting? What happens now? Based on these observations, what do you think affinity means in this simulation?

5. Place both sliders in the Positive Transcription Factor box on the HIGH setting. Predict what will happen to the simulation if you were to move the RNA Polymerase affinity slider to the LOW position. Record your prediction.

6. Now, move the RNA Polymerase affinity slider to the LOW position and record your observations. Was your prediction correct?

7. Place all the sliders in the HIGH position. Check the box to add Negative Transcription Factors and place the concentration and affinity sliders on HIGH. How does this change transcription compared to without Negative Transcription Factors?

Continue to play around with the sliders until you can accurately predict how the change will affect transcription each time.

Additional 3 Questions from the mRNA Simulation:

1. What circumstances make the most mRNA? (What slider positions?)

2. What circumstances make the least mRNA? (What slider positions?)

3. Why would a cell need the option to make or not make a protein?

Part 3: Multiple Cells Simulation

At the bottom of the simulation page, click on the next simulation (it’s greyed out) called Multiple Cells.

Watch the generation of the graph called Average Protein Level vs. Time when one cell is working. If the graph does not automatically begin, then click the Play button at the bottom of the page. Answer the following 4 questions.

1. On the right side of the page, there are controls for Concentration, Affinity, and Degradation. (You need to click the green + to see the sliders.) Predict where you need to place each of the 3 sliders to achieve lots of protein. Record your predictions here:

a. The Concentration slider should be on LOW or on HIGH to achieve lots of protein?

b. The Affinity slider should be on LOW or on HIGH to achieve lots of protein?

c. The Degradation slider should be on LOW or on HIGH to achieve lots of protein?

2. Now, move the sliders into the positions you predicted to see if your predictions were correct. (NOTE: Each time you click “Refresh” to restart the graph, all of the sliders reset themselves to their original setting.) Then, explain why each setting – concentration, affinity, and degradation – makes sense for making lots of protein.

3. Why would a protein need to be degraded?

4. Think back to last week’s lab – Lactase Enzyme Lab. Give an example from that lab of a time when it would be necessary to make a lot of one type of protein.

Part 4: Effects of Mutations on Gene Expression You have learned this week that cells use the two-step process of transcription and translation to transform a protein-coding DNA sequence into a chain of amino acids that makes up a protein. The resulting chain of amino acids will fold into a three-dimensional protein structure that defines the phenotype. Imagine that the following DNA sequence is part of a protein-coding gene. Use this sequence to answer the questions that follow.

… G G A T G C C G C T C T G C A A C T A C…

A) What is the complementary DNA sequence to the DNA sequence above? Hint: look back to your reading and lesson notes to recall the pairing rules for nucleotides A, T, G, and C if you need to!

B) What is the mRNA sequence transcribed from the DNA sequence from Part A? Hint: your answer below should start with the letter G and not C!

C) What corresponding amino acid sequence is translated from the mRNA sequence from Part (B)? Use the genetic code from the lesson or the one posted in the lab. Remember that your amino acid sequence should always start with the START codon!

D) For the following scenarios (i)-(iii), identify the type of mutation that has occurred (single base-pair substitution or frameshift mutation) to our original sequence AND the new amino acid chain that results

from such a mutation. Complete the same sequence from complementary DNA sequence, then mRNA sequence, and then corresponding amino acid sequence like what you did in Parts A, B, and C above!

(i) The 4th C in the original sequence is mutated to a T:

… G G A T G C C G C T T T G C A A C T A C …

Type of mutation:

New amino acid chain:

(ii) An extra C is inserted into the original sequence:

… G G A T G C C G C C T C T G C A A C T A C …

Type of mutation:

New amino acid chain:

(iii) The 5th C in the original sequence is mutated to A:

… G G A T G C C G C T C T G A A A C T A C …

Type of mutation:

New amino acid chain:

E) At the end of translation, an amino acid chain will subsequently fold into a protein with a specific structure and function.

(i) Of the three mutations described in part (D), which mutation will cause the least change to protein function? Briefly explain your reasoning.

(ii) Which mutation would you expect to significantly alter protein function? Briefly explain your reasoning.

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