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DNA Translation.doc

  1. 1. Bio 141 Web Exercise: Translation Lab Objectives: To simulate some pioneering experiments that were used to delineate the genetic code; to study the relationship between the nucleotide sequence of an mRNA molecule and protein synthesis; to demonstrate how a mutation (=change in the nucleotide sequence) existing in an mRNA molecule results in a change in the amino acid sequence of a protein. Readings: Chapter 10 of your textbook Assignments: Follow the instructions given below to produce sequences of ribonucleotides that will be translated into proteins. You will find questions below that you need to answer. Your answers will be handed in during lecture. Be prepared to discuss your answers and how you derived them. IMPORTANT: You are allowed to work in pairs on this exercise. Make sure you equally participate in the exercise---the instructor will have you sign a form that you did. One copy of your notebook and answers to the questions stated below with both your names on it is sufficient. Background: 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. Once you have read and understood the chapter (10) in your book and your lecture notes so that you feel comfortable with the topic, enter the site, press the Start experiment button and begin the exercises listed below. Your website contains a so- called notebook where you can save all your translation exercises. Please save every exercise in your notebook and then save the notebook (e.g. as word or excel file) to print it or send it via e-mail to your instructor. TranslationLab is a web exercise that will allow you to study the importance of the nucleotide sequence of mRNA as the fundamental basis for the genetic code universally deciphered by living cells. You will produce sequences of ribonucleotides that will be translated into protein to simulate the landmark experiments involving cell-free extracts that were essential for interpreting and understanding the genetic code.
  2. 2. Before beginning TranslationLab make sure you are familiar with the concepts described below: 1. The structure of a polypeptide (aminoacids linked by peptide bonds form proteins) 2. The structure and functions of different forms of RNA: --messenger RNA (mRNA): encoded by DNA, scanned by ribosomes to make polypeptides --ribosomal RNA (rRNA): part of ribosomes, helps to make proteins --transfer RNA (tRNA): brings along aminoacids to ribosomes where they will be linked into polypeptide chains 3. The flow of genetic information in a cell: the major processes involved in transcription and translation: DNA to RNA to protein 4. Describe how point mutations (=change of single nucleotide in the DNA) in a gene can affect the amino acid sequence of a protein: these can be a) synonymous (silent), no effect on aminoacids due to redundancy of genetic code (more than one triplett codon codes for the same aminoacid) or b) nonsynonymous: can result in a change from one aminoacid to another, or can result in truncated (=shortened) protein, if a stop codon is introduced. 5. Deletion or insertion mutation: can results in a frameshift so that all nucleotides will be read differently and result in different aminoacids. This is often bad for an organism, since a non-functioning or ill-functioning protein will be made Procedures and Questions This Web exercise is part of Biology Labs Online and you will need an access code to complete the assignment. Instructions will be given in class. First, you need to go to the following website http://biologylab.awlonline.com/index.html. Please select Translation Lab from the right column menu and sign in according to the code your instructor provided. There are specific assignments provided on the webpage. Ignore them and use the assignments provided below instead and answer all the numbered questions printed in bold and italics containted in this document. Hand in your answers to your instructor in class or e-mail them, according to specific instructions you obtained in class. 1. Deciphering The Genetic Code
  3. 3. Because having each of the four nucleotides 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. You will conduct some exercises to test---just as the researchers did originally--- how many nucleotides code for one aminoacid. For that you will start using one nucleotide for each aminoacid and then go up (two nucleotides= a dinucleotide, three nucleotides =a trinucleotide) and try to make protein from your mRNA. 1) Would two nucleotides at a time be sufficient to provide enough codons to code for all 20 amino acids? Why or why not? _________________________________________________________________ _________________________________________________________________ 2) How many amino acids could be coded for by codons containing only two nucleotides? _________________________________________________________________ _________________________________________________________________ 3) 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 (to answer question 1 above). 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. 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
  4. 4. 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. 4) 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. 5)What amino acids do these polynucleotides code for? _________________________________________________________________ _________________________________________________________________ Refer to a codon chart (see chapter 10 in your book). 6) Are the amino acids coded for by the polynucleotides you created consistent with what you would expect based on the codon chart? _________________________________________________________________ _________________________________________________________________ 7) If the code were read two bases at a time, what result would you expect for a polydinucleotide such as AUAUAU? _________________________________________________________________ Try it out and see whether your prediction was correct. 8a) Based on your results, can you say whether the code is even or odd? _________________________________________________________________ 8b) Will you get a different result with UAUAUA than you did with AUAUAU? _________________________________________________________________
  5. 5. This result shows that in these crude extracts translation starts at a random location in the RNA sequence. Translate all possible dinucleotides with TranslationLab. 9a) Did you get all of the amino acids? ____________ 9b) If not, which ones are missing? ____________________ 9c) Did you get any amino acids more than once? ______________ 9d) Which ones? ______________ 9e) What does this tell you about the code? ____________________________ 10) From what you have already discovered, what do you think will happen if you use a polytrinucleotide such as AGC? ________________________________________________________ Try it out. 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. 11a) Did you get the result you expected? Explain what happened. _________________________________________________________________ _________________________________________________________________ 11b) Will ACG or GCA give a different result? _________________________________________________________________ 12a) From these results, can you now tell how many bases there are in a codon? ___________________________
  6. 6. 12b) If so, how many are there and how do you know this? _________________________________________________________________ 12c) 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? _________________________________________________________________ 12d) If so, which codon and which amino acid go together? _________________________________________________________________ 12e) By elimination, can you assign another codon-amino acid pair? What is it? _________________________________________________________________ Try CAC next. 13a) Did the results support your codon assignment? ________________ 13b) Is there evidence here that one of the amino acids must have more than one codon that codes for it? If so, which one? _________________________________________________________________ Try out further different combinations, changing only the last letter. Confirm your results by referring to the codon chart in your book (Ch.10). 14) Referring to the codon chart, what is the minimum and maxium number of codons coding for one amino acid?
  7. 7. _________________________________________________________________ Use tetranucleotides to figure out which amino acids go with the codons that can be produced using only A and U. 15) What unusual result did you see with some of the tetranucleotides and what is your explanation for this result? _________________________________________________________________ 2. Altering the Genome: 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 point mutation creates an altered form of hemoglobin that produces the genetic disorder called sickle-cell disease (sickle-cell anemia). The purpose of the following assignment is to demonstrate the effect a single point mutation can have on the amino acid sequence of a protein. You will learn more about the inheritance of sickle cell anemia in future genetics classes. Here the disease serves as a real life example of an inherited disorder in humans caused by only one nucleotide change in the DNA. 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. 1a) Synthesize a mRNA from the trinucleotide sequence GAA. Enter this sequence in your notebook. 1b) Translate this mRNA and enter the results in your notebook. _________________________________________________________________ 1c) Synthesize and translate the trinucleotide GUA and do the same for the trinucleotide GAG. _________________________________________________________________ 1d) 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.) _________________________________________________________________ 2) What amino acid does the codon GAA specify? 3) Which amino acid is incorporated into the sickle-cell hemoglobin molecule when this codon is mutated to GUA?
  8. 8. _________________________________________________________________ Perform other experiments if necessary to confirm your codon assignments to answer this question. 3. Point Mutation 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,often even across different kindgoms. 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 (=more than one codon codes for the same amino acid) 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 in many vertebrate species. As a result of this conservation of nucleotide sequence, the amino acid sequences will be almost identical. Comparing the amino acid sequence for insulin from cows, humans, sheep, dogs, and rats often shows fewer than six or seven differences. However, point mutations (see background # 4 and exercise 2. above) in certain positions of a codon can create changes that dramatically alter the aminoacid sequences and therefore the protein produced. Examples of mutations of this type include frameshift mutations (see background # 5). To help you understand why the nucleotide sequences for important genes are highly conserved, 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 an 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
  9. 9. 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. G) Summary The exercises above were designed to help you become familiar with the genetic code and the mutations that can affect the genome of any organism. You hopefully realized that the genetic code is universal among organisms and that it has to be very precise. Even minor changes in the nucleotide sequence (mutations) can have drastic effects on the organism, since a change in DNA ( e.g. one more (=insertion) or one less (=deletion) nucleotide will be transcribed into a different mRNA and can result in a different amino acid or change all amino acids of the protein (e.g. a frameshift mutation). We all accumulate a number of mutations in our genomes over our lifetime; luckily most of these have no effect on our health at all---they are either silent mutations without an effect on the amino acid outcome or are located in parts of the genome not encoding any proteins, or we end up with one good and one bad copy of a gene on our two chromosomes. However, some mutations may have an effect on our offspring since the offspring may end up inheriting the chromosome with the “bad” copy of a gene from you. To better understand how mutations can affect an organisms and future generations, it is important for you to understand the very basic principles of inheritance (covered extensively in the Genetics lab modules of this course) and of the genetic code. Please keep in mind that the principles outlined here apply to plants as much as to animals and other organisms.