Just one nucleotide! Exploring the effects of random single nucleotide mutations

Transcription

1 Dr. Beatriz Gonzalez In-Class Worksheet Name: Learning Objectives: Just one nucleotide! Exploring the effects of random single nucleotide mutations Given a coding DNA sequence, determine the mrna Based on the transcribed mrna, predict the resulting protein sequence Become proficient in using the codon chart to determine which codons code for which amino acids Recognize the importance of the START and the STOP codons Predict the effect that different types of mutations will have on the protein expressed Introduction The Central Dogma of molecular biology is the concept that cells are ruled by a cellular chain of command that flow from DNA to RNA to protein. The information is housed in the DNA, transcribed into a RNA molecule and translated into a protein. Therefore, protein synthesis occurs by means of transcription and translation. Transcription occurs in the nucleus and produces RNA pairing complementary bases to the coding DNA strand. This RNA is modified and processed in eukaryotes and then goes to the cytoplasm where translation takes place (either in polyribosomes or in ribosomes attached to the rough endoplasmic reticulum). Here the mrna code is converted into a protein, a chain of amino acids. Activity 1: Review of base-pair rules 1. Fill out the following table to review the complementary base-pair rules during the processes of DNA replication vs. transcription: DNA Replication If DNA template has Newly made DNA this base: will have: A C G T Transcription If DNA template has mrna will this base: have: A C G T

2 Activity 2: Transcription: Making mrna In this simple exercise, you will transcribe the coding DNA sequence into an mrna. This particular region does not contain introns. 1. Using the following coding DNA sequence, determine the resulting mrna. DNA sequence: (provided sequence shown 3 5 ) nt = nucleotide # nt T T C A T A C G A C G T C T A C G T A A C T G C T mrna sequence: (5 3 ) Activity 3: Translation: mrna Universal Codon Chart Translation is the process that takes the information that was passed from DNA into the messenger RNA and turns it into a linear sequence of amino acids covalently joined by peptide bonds. It really is a translation from one code, nucleotide sequence, to another code, amino acid sequence. 1. Name the three different types of RNA. Mention their functions. 2. Where are ribosomes made and what are they made of? How do they get to their final destination?

3 3. Refer to the universal codon chart below. The AUG codon, codes both for methionine and serves as an initiation site; the first AUG in an mrna's coding region will be the site where translation (not transcription) into protein begins. Universal Codon Chart Table 1 : Codon table. This table illustrates the 64 possible codon triplets. 2nd base U C A G UUU Phenylalanine UCU Serine UUC Phenylalanine UCC Serine U UUA Leucine UCA Serine UUG Leucine UCG Serine UAU Tyrosine UAC Tyrosine UAA Stop UAG Stop UGU Cysteine UGC Cysteine UGA Stop UGG Tryptophan CUU Leucine CUC Leucine C CUA Leucine 1st base CUG Leucine AUU Isoleucine AUC Isoleucine A AUA Isoleucine 1 AUG Methionine GUU Valine GUC Valine G GUA Valine GUG Valine CCU Proline CCC Proline CCA Proline CCG Proline CAU Histidine CAC Histidine CAA Glutamine CAG Glutamine ACU Threonine AAU Asparagine ACC Threonine AAC Asparagine ACA Threonine AAA Lysine ACG Threonine AAG Lysine GCU Alanine GCC Alanine GCA Alanine GCG Alanine GAU Aspartic acid GAC Aspartic acid GAA Glutamic acid GAG Glutamic acid CGU Arginine CGC Arginine CGA Arginine CGG Arginine AGU Serine AGC Serine AGA Arginine AGG Arginine GGU Glycine GGC Glycine GGA Glycine GGG Glycine a) What is a start codon? How many are there? Name the codon and amino acid: b) Name all the amino acids that have only one codon: c) Name all the amino acids that have multiple codons: d) What does it mean when a codon translates into STOP? Name the three STOP codons.

4 Activity 4: Translation: Determining the Protein Sequence Using the following coding DNA sequence, determine the mrna and the polypeptide chain coded by this region of the DNA. (You determined this sequence earlier in activity 2) DNA sequence: (provided sequence shown 3 5 ) nt = nucleotide # nt T T C A T A C G A C G T C T A C G T A A C T G C T mrna sequence: (5 3 ) (the same as in activity 2) Amino acid sequence (polypeptide): Activity 5: Exploring Point Mutations Procedure: 1. Mutate nucleotide #10 in the DNA sequence provided according to the rules in table Your instructor will provide you with rubber dice so you can simulate what happens when the DNA sequence is mutated if the number you roll does not result in a change, keep rolling until it changes. 3. Every student in your group should roll the die to mutate their own DNA. In this way, there will be more outcomes to compare. 4. Write down your newly mutated DNA sequence. 5. Determine and record the mrna and the protein sequence coded by the mutated DNA sequence. Table 1: Rules for determining which kind of mutation will take place If you toss a Then you must 1 Substitute your nucleotide with an A 2 Substitute your nucleotide with a C 3 Substitute your nucleotide with a G 4 Substitute your nucleotide with a T 5 Delete the nucleotide 6 Insert a nucleotide right after (toss again until you get 1 4 to determine which letter nucleotide to insert)

5 DNA sequence: (provided sequence shown 3 5 ) nt = nucleotide # nt T T C A T A C G A C G T C T A C G T A A C T G C T Write complete new DNA sequence with mutation at nucleotide 10 position: (Depending on the mutation, you will end up with a 24-nt, 25-nt, or 26-nt DNA sequence.) mrna sequence from mutated DNA: Amino acid sequence (polypeptide): Circle any differences from original protein Activity 6: More Mutations! Keeping the mutated nucleotide, mutate nucleotide #16 following the procedure outlined in activity 5. Write the resulting DNA, mrna, and protein sequences Write complete DNA sequence (now with two mutations: one at nt 10 position, and the second at the nt 16 position): mrna sequence with both mutations: Amino acid sequence (polypeptide): Circle any differences from original protein

6 Activity 7: Thought Questions 1. Write the definition of the term mutation. 2. If a mistake is made during transcription, will that mistake be permanent? During translation? Why or why not? Will it be passed on to the next generation of cells? Explain your answers. 3. Why do you think you used a die to determine the mutations? Why did you keep the first mutation when mutating the sequence a second time? In other words, why didn t you fix it? 4. Did everyone in your group get the same mutation(s)? Explain. What are the odds of two people rolling the same number? Of rolling the same mutation? 5. Deletions and insertions are also called - mutations. 6. What is a point mutation? Differentiate between the following types of point mutations: a) Substitution b) Deletion c) Insertion d) Missense e) Nonsense 7. Explain the statement: "A mutation causes a change in the genotype, but that change does not always cause a change in the phenotype". Reference: Gonzalez, B.Y. and J. H. van Oostrom Using Dice to Explore the Consequences of DNA Mutations, Journal of College Science Teaching 38 (5): 56-59