Human Hemoglobin Activity
Adapted from www.biochem.mcw.edu
Background: This activity allows you to look at the actual nucleotide sequence of the beta-globin protein subunits that make up the protein hemoglobin. Hemoglobin is composed of two alpha-subunits linked with two beta-subunits (basically, four subunits of proteins called a tetramer). Each subunit binds a heme group, which in turn binds one iron atom( Fe). Oxygen can bind to this Fe atom. As a red blood cell passes through the lungs, oxygen diffuses into the cell and binds to the Fe of the heme group on all four subunits of the hemoglobin tetramer (four subunits in the protein). Sickle cell anemia is caused by a single point mutation in the nucleotide sequence of beta-globin. The mutation is located in the seventh codon. The seventh codon should read GAG (which codes for the amino acid glutamic acid), but the middle nucleotide has changed to a thymine, which changes the codon to GTG (which codes for the amino acid valine). Normally glutmatic acid, which is hydrophilic, is placed on the outside of the protein, thus coming in contact with the water environment of the blood. However, when the mutation occurs, valine, which is hydrophobic, moves to the inside of the protein. This single change results in a sickle shape that is not able to carry as much oxygen, leading to the symptoms of the Sickle Cell Anemia.
We will only be looking at a portion of the complete hemoglobin protein, there are actually 73,308 nucleotides that code for the hemoglobin gene. This gene is located on the tip of chromosome 11 and represents only about 0.002% of the entire human genome. You will only be looking at the beta-globin sequence that is located between nucleotides 62,187 to 63610. You are going to trace the flow of information from DNA to mRNA and finally to the sequence of amino acids that comprise the beta-globin protein.
Keywords: hemoglobin, beta-globin, heme, Fe, nucleotide, codon, DNA, exon, intron, gene, spliceosome (enzyme), mRNA, transcription, translation, protein synthesis, protein, mutation.
1. You will be given several pages of the actual nucleotide sequence of beta-globin. Look them over. The bottom strand of nucleotides "the Crick strand" is the non-coding strand or template strand (the top line the "Watson" is equivalent to the actual genetic code written in DNA.).
2. There are actually, 1,423 nucleotides in the DNA for beta-globin, enough to code for 474 amino acids. However, beta-globin protein is only known to have 146 amino acids. What is all the excess DNA doing? It is serving as “introns” that separate the protein-encoding “exons”. The beta-globin gene consists of three exons and two introns:
Exon #1 62187-62278
Intron #1 62279-62408
Exon #2 62409-62631
Intron #2 62632-63481
Exon #3 63482-63610
Locate the introns and exons using a pencil, put brackets around them as follows: G)(GGGGGGGGGG))(GGGG. Double and triple check your work with other students. Label the exons and introns. Only after you have done this, highlight the exons only and cross off the introns so you know which sequences you will eventually need to discard.
3. Transcribe the exons only into mRNA by writing the complementary mRNA next to the "Crick" DNA base sequence (use a slash mark every three nucleotides to avoid visual confusion).
4.. Cut out the DNA base sequences and tape together into a long strip.
5. Imitating the role of a spliceosome, splice out the introns by folding the paper between the introns and exons and then cutting out the introns.
6. Bond the exons, by taping them together; this is the code that will leave the nucleus for the formation of a protein. Staple the completed mRNA into your journal (take great care not to cut out any nucleotides that are part of an exon).
7. When complete, attach a Guanine cap (draw a "G" on the front of the sequence and add a poly-Adenine (up to 200) tail to the mRNA (be creative, make your own). This is now ready to leave the nucleus and move into the cytoplasm.
1. Starting with the first exon on the mRNA, identify each codon by placing a slash mark between each codon, like this:
2. Using your codon-amino acid chart (there is an online version at Additional Information for this activity ), translate the mRNA into a protein chain. Write the name of each amino acid under each codon (use the three letter abbreviation, example alanine "ALA".
Post Lab Questions: Be as specific and detailed as you can.
1. What is the seventh DNA codon? Write it out along with the appropriate amino acid. Now, write the mutated DNA codon sequence for Sickle Cell Anemia along with the corresponding amino acid above the regular amino acid (highlight the codon).
2. Define the key terms (located below the introduction paragraphs).
3. What is the role of RNA polymerase?
4. Besides trimming the introns out of mRNA, what else must be done to a mRNA before it can successfully leave the nucleus?
5. What part does the ribosome and transfer RNA play in translation?
6. What is the role of the spliceosome?
7. How do mutations affect the final protein product? What are all the possible ways a mutation can affect the function of the protein?
8. How is this activity related to the Central Dogma?
9. Look up Sickle Cell Anemia. What are the symptoms? What is the link to malaria? (you must include a bibliography)
10. What are three ways mistakes can be made during transcription and translation? How are those mistakes similar to a mutation in the DNA? (hint: think of the final protein product)
Additional Information for this activity