Assignments for majors

During the late 1950s and early 1960s researchers were able to solve one of the major secrets of life: how genes worked. The problem the researchers were trying to solve was how a linear sequence of four nucleotides (A, G, C, and U) determined the amino acid sequence of proteins, which were made out of up to 20 different amino acids. The following assignments are designed to help you reproduce some of the experiments that the scientists used to figure out how this was accomplished. Your mission, should you choose to accept it, is to crack the genetic code of life.

Assignment 1
The Genetic Code

A major step forward in figuring out the code was the discovery by Nirenberg in 1961 that a cell-free extract made from E. coli cells could translate RNA added to the extract into proteins. The composition of the newly synthesized proteins could be determined by measuring the incorporation of radioactive amino acids into these proteins as they were translated. In his first experiment he made poly U RNA, using the enzyme polynucleotide phosphorylase, and translated it into a peptide of polyphenylalanine using the cell-free extract. This was definitive proof that RNA could code for the synthesis of proteins and gave the first possible assignment of a nucleotide code to the amino acid it specified.

  1. Because having each nucleotide code for only one amino acid would allow for only four different amino acids to be incorporated into a protein, it was obvious to researchers that there had to be a conversion between multiple bases and each amino acid. Would two nucleotides at a time be sufficient to provide enough codons to code for all 20 amino acids? Why or why not? How many amino acids could be coded for by codons containing only two nucleotides? Will three nucleotides per codon work? Why or why not? Explain your answers. To answer these questions using TranslationLab, click the Start Experiment button on the input screen of TranslationLab. For each of the four bottles of ribonucleotides that appear, click on the arrow to select a nucleotide. Do this for two nucleotides initially. Click the Make RNA button to display the sequence of mRNA that you created. Click Add to Notes to create a record of your experiment. To translate this sequence into amino acids, click on the To Translation Mix button. Click Add to Notes to add the amino acid sequence to your notebook. Continue this process until you are able to answer the questions above.

  2. Once it was determined that codons consisted of three-nucleotide sequences, the specificity of each codon could be determined. Use TranslationLab to determine what poly U codes for by performing the following exercise. Click the Start Experiment button on the input screen of TranslationLab. For each of the four bottles of ribonucleotides that appear, click on the arrow to select the uracil (U) nucleotide. Click the Make RNA button to display the poly U sequence of mRNA that you synthesized. Click Add to Notes to create a record of your experiment. To translate this sequence into amino acids, click on the To Translation Mix button. What does poly U code for? Click Add to Notes to add this peptide sequence to the poly U sequence. Repeat the same procedure to make polynucleotides of each of the other three nucleotides. What amino acids do these polynucleotides code for? Refer to a codon chart (see Campbell, N. A. Biology 5/e, chapter 17). Are the amino acids coded for by the polynucleotides you created consistent with what you would expect based on the codon chart?

  3. Although the Nirenberg experiments showed that RNA did determine the amino acids in the protein, they did not show how many bases were used for each codon, whether the codons were overlapping (is the second codon read from the second base of the first codon [overlapping] or from the first base after the last base of the first codon? [no overlap]), or whether there could be bases in between the codons that did not code for anything (AUCGGGAACGGGACAGGGG, for instance, where the G's in between AUC, AAC, and ACA aren't translated into amino acids­just as we use spaces to separate words in a sentence). Khorana developed a means to produce polydinucleotide and later, polytrinucleotide and polytetranucleotide sequences of DNA that could then be transcribed into RNA to be added to the cell-free translation mix.

  4. If the code is read two bases at a time, what result would you expect for a polydinucleotide such as AUAUAUAU? Try it and see whether your prediction was correct. From your results can you say whether the code is even or odd? Will you get a different result with UAUAUAUA than you did with AUAUAUAU? This result shows that in these crude extracts translation starts at a random location in the RNA sequence. Translate all possible dinucleotides that use two different bases with TranslationLab. Did you get all of the amino acids? If not, which ones are missing? Did you get any amino acids more than once? Which ones? What does this tell you about the code?

  5. From what you have already discovered, what do you think will happen if you use a polytrinucleotide such as AAC? Try it. To help analyze your results, once you have entered the polytrinucleotide sequence, click the Make RNA button and then add this sequence to your notebook by clicking the Add to Notes button. Add the peptide sequence produced from this RNA to your notebook as well. Did you get the result you expected? Explain what happened. Will ACA or CAA give a different result? From these results can you now tell how many bases there are in a codon? If so, how many are there and how do you know this? Comparing this result with the result from polydinucleotide AC, can you now specify a codon for one of the amino acids incorporated by these templates? If so, which codon and which amino acid go together? By elimination, can you assign another codon­amino acid pair? (Hint: Using what you know now, look back at the dinucleotide experiment with AC.) What is it? Try CAC next. Did the results support your codon assignment? Is there evidence here that one of the amino acids must have more than one codon that codes for it? If so, which one? Confirm your results by referring to a codon chart.

  6. What do you think will happen if you translate a tetranucleotide? Try translating the tetranucleotide CAAG. Did you get the result you expected? Can you now assign a codon to any of the other amino acids that appeared in problem 5? (Don't worry about any new amino acids that showed up here, just solve the codons for the amino acids in problem 5.) If so what are they? Test your assignment with AACG. Did this confirm your results? Using the above data and any other experiments that are necessary, assign amino acids to all possible codons that do not include G or U, only various combinations of A and C.

  7. Now try AU, AAU, and AUU. Did you notice something different this time? What happened, and how would you explain this unusual result? List any new codon assignments that you were able to make from these experiments. Use tetranucleotides to figure out which amino acids go with the codons that can be produced using only A and U. What unusual result did you see with some of the tetranucleotides and what is your explanation for this result?

  8. Now try GGG, GGA, GGC, and GGU. What amino acid showed up in all four experiments? Are there any codons shared in common by these four reactions? If not, then what must be true to explain your results? Can you propose a codon or codons for the amino acid that showed up in all four experiments? Do the codons that you've just assigned to this amino acid have anything in common? What is it? Use tetranucleotides to prove that your assignment is correct. Comparing these results with the ones above, can you say whether some positions in the codon are less important than others in specifying which amino acid is coded for?

Assignment 2
Altering the Genetic Code: Mutations

Single nucleotide changes (point mutations) in the sequence of a gene can result in changes in the amino acid sequence of a protein produced from the mutated gene. One of the most well studied examples of the effects of a mutation on the sequence of a protein involves the oxygen-transporting protein hemoglobin. A mutation creates an altered form of hemoglobin that produces the genetic disorder called sickle-cell disease (sickle-cell anemia). You will learn more about hemoglobin and the effects of mutations in the hemoglobin gene in HemoglobinLab. The purpose of the following assignment is to demonstrate the effect of a point mutation on the amino acid sequence of a protein.

  1. Sickle-cell disease results from a point mutation in the second nucleotide of the codon GAA, which results in a change in the amino acid at position 6 in the hemoglobin protein. Synthesize a mRNA from the trinucleotide sequence GAA. Enter this sequence in your notebook. Translate this mRNA and enter the results in your notebook. Synthesize and translate the trinucleotide GUA and do the same for the trinucleotide GAG. Assign codons to each amino acid produced from the three mRNA sequences. (Hint: consider what you know about the sequences for stop codons from attempting to assign codons for each amino acid.) What amino acid does the codon GAA specify? Which amino acid is incorporated into the sickle-cell hemoglobin molecule when this codon is mutated to GUA? Perform other experiments if necessary to confirm your codon assignments to answer this question.

Assignment 3
Group Exercise

Because the genetic code is a universal code in biology, in general the nucleotide sequence of important genes is highly conserved across many different species of organisms. It is very common for 70% or more of the nucleotides in a gene to be conserved among very different organisms. Redundancy in the genetic code allows for small differences in the nucleotide sequence for a given gene without significant variations in the amino acid sequence of a protein. For example, the nucleotide sequence of the gene for insulin, the peptide hormone required for glucose uptake by many body cells, is well conserved (greater than 80% similarity) in many vertebrate species. As a result of this conservation of nucleotide sequence, comparing the peptide sequence for insulin from cows, humans, sheep, dogs, and rats often shows fewer than six or seven differences in amino acid sequence. However, point mutations in certain positions of a codon can create changes that dramatically alter the protein produced. Examples of mutations of this type include frameshift mutations. To help you understand why the nucleotide sequences for important genes are highly conserved, work together in a group of four or five students to complete the following assignment.

Imagine that you have just purified a new protein from the brain of adolescent males that you believe may be responsible for excessive hair-combing behavior. From peptide-sequencing experiments, you have determined that this protein contains the following peptide sequence: Trp-Met-Asp-Gly-Trp-Met. Determining the nucleotide sequence of mRNA that was used to translate this part of the protein will enable you to identify the chromosomal location of this new gene and allow you to isolate and clone this gene. It is known that this peptide sequence is highly conserved among males that demonstrate excessive hair-combing behavior which suggests that this portion of the protein is important for its functions. In addition, a mutant form of this protein has also been discovered that appears to result in the loss of the excessive hair-combing behavior. This mutant sequence arises from a single point mutation in the nucleotide sequence of the normal (wild-type) gene for this protein that creates a frameshift mutation. The peptide sequence from this mutant protein is Met-Tyr-Val-Cys-Met- Tyr. Use TranslationLab to complete the following exercises.

  1. Determine the sequence of a mRNA that could be used to translate this peptide.
  2. Can you determine another sequence of mRNA that would also code for this peptide? Why or why not? Explain your results.
  3. Once you have deciphered the mRNA sequence for the normal protein, introduce changes in this sequence until you have determined the nucleotide sequence that specifies the mutated peptide sequence. Examine this mRNA sequence and identify the codon or codon(s) that were altered to create the mutant peptide.