Bio200 Cell Biology Activity – ********KEY******** Autumn 2014

Model 1: Elongation Phase of Translation

1. Label as many components of the cartoon as you can.

2. Label the 5' and 3' sides of each codon and anticodon.

3. a. How many nucleotides are there in a codon? 3 In an anticodon? 3

b. Do codons overlap? no

c. Which molecule contains codons? mRNA Which contains anticodons? tRNA

d. How many amino acids does each tRNA carry? 1

4. The following statements are partially correct. Write a better statement for each one that is completely correct and describes the process more accurately.

a) The codons bind to anticodons and then the anticodon makes a . Codons form hydrogen bonds with the anticodons of tRNA molecules. Then the catalyzes the formation of peptide bonds between the amino acids being carried by the tRNAs.

b) If the anticodon is attached to the wrong , then the ribosome will still use the anticodon but no protein will be formed. If the tRNA that has a certain anticodon is attached to the wrong amino acid, the ribosome will have no way of knowing this. It will simply attach that amino acid to the growing protein.

5. Translation ALWAYS begins with a tRNA carrying the amino acid Met. a. What is the sequence of the ""? Label with 5' and 3'. 5'AUG3'

b. In which direction must the ribosome move ("translocate") along the mRNA? 5' 3' (From 5' to 3' or from 3' to 5'?). How did you decide this?

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Model 2: Initiation Phase of Translation

The ribosome is made up of both protein and RNA. A small ribosomal RNA is positioned in the small subunit that will bind to a part of the mRNA.

6. Label as many components of the cartoon as you can.

7. a. Circle the ribosome binding site (RBS) in the mRNA.

b. Is the RBS closer to the 5' or 3' end of the mRNA? 5'

c. Which are more prevalent in the Ribosome Binding Site, pyrimidines or purines? purines

d. What types of bonds hold the mRNA and small ribosomal subunit together? hydrogen bonds

e. What other molecules (in this figure) are held together with these same types of bonds? the mRNA and the tRNAs (H-bonds of base pairs)

8. a. Does the first tRNA bind before or after the ribosome is complete? before

b. Translation ALWAYS begins with a tRNA carrying the amino acid Met. What is the name of the sequence that that first tRNA binds to? start codon

9. a. How many nucleotides are there between...

... the RBS and the start codon? 4

... the 5' end of the mRNA's and the start codon? 14

b. Are either of your answers in "a" multiples of 3? no!!!

c. Using what you know from 9a and 9b, what establishes the "frame" of triplet codons for translation? the start codon "sets the frame" 10. In the mRNA sequence below, circle and label the most likely RBS and the start codon. Note: Like many nucleotide sequences, the RBS can vary between species

5' UCUUAAGAAGGAUCUGUAAUGUCUGUAUGUCUGUAGUGUAUGUCUUGUAUCG 3'

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Model 3: Termination Phase of Translation

"release factor"

11. Label as many components of the cartoon as you can.

12. List two things that are different between the release factor and a tRNA: release factor does not have an amino acid attached, it doesn't have an anticodon, not a nucleic acid

13. List two things that happen after release factor binds to the ribosome: i. the new protein is separated from the last tRNA ii. the two ribosomal subunits separate and the tRNAs, the mRNA and the release factor dissociate

14. What is the sequence of the codon that indicates the end of this protein? 5' UAG 3' (This is called a "".)

15. Is release factor an enzyme? Yes What is your reasoning for your answer? After release factor binds, the covalent bond between the new protein and the last tRNA is hydrolyzed. A catalyst is needed to break a covalent bond, and release factor is a protein, therefore it is an enzyme.

16. What would happen if there were a tRNA in the cell that could base pair with a stop codon? Then it would compete with release factor for binding to stop codons, meaning that translation would not always stop at a stop codon, and those proteins would be longer.

17. The release factor binds to the A-site of the ribosome (the one on the right) relatively weakly compared to the correct tRNA. Why is this weak binding a good thing for correct translation of proteins? Release factor is forming hydrogen bonds with the specific bases of the stop codons. If it was able to bind more strongly, or bind non-stop codons… then it would possibly terminate translation too often, resulting in shorter proteins.

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Integration Questions Learning goal: synthesize information from all three phases of translation with your new group

General questions for all group members 1. What is the purpose of translation? To synthesize proteins using an mRNA template

2. Orient yourself to the schematic on the right. Identify the following: f-Met, tRNA, mRNA, ribosome and anticodon.

3. In the chart below, fill in the function of each of the components and whether they are necessary for the different stages of translation

Component Function Necessary for Necessary for Necessary for initiation? elongation? termination? mRNA Template for synthesis, Yes Yes Yes binds ribosome and tRNAs f-Met tRNA Binds to start codon Yes No (except for No the first peptide bond) ribosome "Platform" and catalyst for Yes Yes Yes translation release Binds stop codons and No No Yes factor catalyzes hydrolysis of protein from last tRNA anticodon Part of a tRNA that binds to Yes Yes No (except for the codon in an mRNA the tRNA holding the peptide in the P-site

------Initiation Integration Questions: Initiation Expert helps other group members answer the following questions.

1. Imagine you replace four purines with pyrimidines in a ribosome binding site. Which of the following statements would be true? F The rate of formation of peptide bonds will decrease T The affinity of the small ribosomal subunit for ribosome binding site will decrease T The rate of initiation will decrease F The affinity of large ribosome subunit for small ribosome subunit will decrease

2. What establishes the ? (circle the correct answer) A. The ribosome binding site B. The start codon C. The 5’ end of the mRNA D. The distance between the ribosome binding site and the start codon

3. What is the next codon that will be read by the ribosome in the schematic above? 5' GAA 3'

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4. Stop and reflect. All group members should now be able to explain 1) how the reading frame is established, 2) the purpose of the ribosome binding site and 3) the triplet nature of codons.

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Elongation Integration Questions: Elongation Expert helps other group members answer the following questions

1. A covalent bond between which two molecules keeps the growing protein chain attached to the ribosome? a tRNA and the last amino acid of the growing protein

2. Which direction will the ribosome move in the model shown above? to the right

3. What is the function of the tRNA? The tRNA binds to a codon and carries the corresponding amino acid.

4. What would happen if all the tRNAs in a cell with anticodons for lysine carried glycine amino acids instead of lysine amino acids? Circle the correct answer. A. Translation would still occur but the protein product would contain glycines in place of lysines B. Translation would stop at the first lysine codon C. Translation would still occur but there would be no lysines or glycines in the protein D. Translation would still occur but the protein product would contain lysines in place of glycines

5. Stop and reflect. All group members should now be able to explain 1) the relationship between codons and anticodons, 2) how the structure of a tRNA impacts its function and 3) why the ribosome moves in a 5’ to 3’ direction.

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Termination Integration Questions: Termination Expert helps other group members answer the following questions

1. Identify the release factor in the model to the right 2. Compare and contrast a release factor to a tRNA: How are they different? How are they similar? Different: Release factor doesn't carry and amino acid, it is made of protein, and does not have an anticodon (it cannot "base pair" with the mRNA)

Similar: The overall shape of a tRNA and release factor is similar so they both bind in the tRNA binding site of the ribosome, they both bind to codons.

3. What has to happen for the growing protein to be released from the ribosome? (Hint: look back at Elongation Integration Question 1) The bond between the last amino acid and the tRNA has to be broken.

4. How does the structure of the release factor impact its function? The shape is important for fitting in the tRNA binding pocket, and the "bottom" part must form hydrogen bonds with the bases in stop codons so that it will stay long enough to hydrolyze the bond.

5. Stop and reflect. All group members should now be able to explain 1) the role of a release factor during termination and 2) how the structure of the release factor impacts its function. ------

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On your own questions for practice:

The reaction catalyzed by the ribosome is shown to the right. amino acid

1. Label an arrow pointing to...... an "Amino-acyl tRNA" tRNA with the ... the "Peptidyl tRNA" nucleotide at one end ... the "Empty tRNA" shown much larger than the rest of the molecule 2. Which end of the tRNA is attached to the amino acid, 5' or 3'? 3'

3. a. In the top half of the model...

... circle the two atoms that will be connected by a new bond.

... draw a slash through the bond that will be broken. b. In the bottom half of the model, circle the newly formed bond. N

2. The drawing to the right shows a short protein of 8 amino acids that is complete, but is still in the ribosome. a. Label the amino terminus and the soon-to-be-carboxyl terminus of the protein. b. Circle the bond that needs to be broken before the protein can be used. c. What molecule catalyzes the reaction in "b"? ______d. Draw a square around a peptide bond.

C

7 Bio200 Cell Biology Activity – Translation Summer 2014 3. This is the sequence of a complete mRNA from a bacterial cell:

5' UCAAGGAGGCGUUAGCAUGAAAUUUAUGGGGCGGGUAUAGCUAGCAUUUCAAG 3' a. Write the protein sequence that is translated from this mRNA on the line below, and label the amino (N) and carboxyl (C) termini of the protein.

N-Met-Lys-Phe-Met-Gly-Arg-Val-C b. How many tRNAs will bind to the ribosome to make this protein? 7 c. Which of the following sequences within the mRNA most likely contains the ribosome binding site? (Circle ONE)

5'AAGGAGGC3'

4. For each different mutant cell described below, assume that ONE specific molecule or part of a molecule is mutated in that cell so that the molecule’s function has changed. Name as many molecules that could result in the description (but remember that for the mutant phenotype, you are considering each mutation by itself).

Cell 1: In many different types of proteins, there is the amino acid Thr (threonine) where an Ala (alanine) should be.

The amino-acyl tRNA synthetase that normally binds alanine now binds threonine instead

Cell 2: Many different types of proteins are much shorter than in a normal cell, but have the correct sequence up to that point. tRNA levels are normal in the cell. release factor

Cell 3: About a third of all new proteins in a mutated cell are not doing their jobs correctly. When you compared to proteins in a healthy cell, these proteins appear much larger overall.

Some tRNA has changed it’s anticodon to recognize one of the three STOP codons, so this is erroneously allowing elongation to continue in proteins that normally use that STOP codon.

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