Pauling/Itano Experiment

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1 Chapter 12

2 Pauling/Itano Experiment Linus Pauling and Harvey Itano knew that hemoglobin, a molecule in red blood cells, contained an electrical charge. They wanted to see if the hemoglobin in normal RBC s is different than the hemoglobin in sickle RBC s. To do this, they compared an electrophoresis experiment of the two hemoglobin structures with known hemoglobin samples

3 Pauling/Itano Experiment Based on this experiment, they found that RBC s contain a type of hemoglobin called HB A, while Sickle RBC s contain HB B. Later, they compared the polypeptide (amino acid) chains of each strand of hemoglobin They thought the two chains would be completely different. Instead they were surprised to find the difference between a normal and sickle RBC gene sequence is only one amino acid out of 438. One amino acid went wrong, and it completely changed the protein

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6 RNA RNA is a polymer of nucleotides. Only a few differences exist between DNA and RNA RNA contains uracil instead of thymine RNA is generally single-stranded RNA freely leaves the nucleus of cells RNA strands can pair with DNA strands according to the same base-pairing rules. So RNA serves as an excellent copying system for DNA. C pairs with G whether its DNA or RNA A (DNA or RNA) pairs with T (DNA) or U (RNA)

7 RNA There are hundreds of classes of RNA, but we care about three of them Messenger RNA (mrna): takes a message from the nucleus to the ribosome Transfer RNA (trna): carries amino acids to ribosomes Ribosomal RNA (rrna): makes up a portion of ribosomes

8 Transcription Transcription is the process of forming an mrna strand as a copy of a DNA strand. The purpose is to build a specific protein using a gene sequence. This occurs separately and independently from replication 1. The DNA strand unwinds and unzips similarly to replication. The location this occurs is called a promoter. Promoters are sequences of DNA that identify the origin of a gene sequence. The strand will continue to unwind until it reaches a sequence called the terminator.

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10 Transcription 2. Once the strand unwinds, an enzyme called RNA polymerase attaches RNA nucleotides in the 5 3 direction only. 3. When the polymerase reaches the terminator, it stops and releases an RNA transcript called mrna. 4. The mrna then exits the nucleus to be picked up by a ribosome. The cell will have multiple RNA polymerases working simultaneously to ensure the maximum output of mrna sequences.

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13 Introns and Exons To enhance the integrity of the mrna strand when it leaves the cell, a couple steps occur First, a cap is put on the 5 and 3 ends of the strand so that nothing can accidentally break off or be added to the strand. Second, sections of the mrna are removed by a spliceosome The sections that are removed are called introns. The remaining sections, called exons, are what get expressed (read) by ribosomes

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15 Introns and Exons Why introns and exons? The short answer is, who knows? Introns create redundancy, which helps reduce the likelihood that a mutation will cause a problem If 100% of the nucleotides are expressed, then a mutation will cause a problem 100% of the time. Introns allow for more efficient gene sequences Take the word hearth. From this word, you get he, ear, art, heart, and earth, depending on which letters you cut out.

16 Translation Translation is the process of going from a strand of mrna to a strand of amino acids. For this to occur, you need a trna molecule. trna is a molecule built from an RNA strand. Bound to one end of the trna is a specific amino acid. Bound to the opposite end of trna is a sequence of three nucleotides called an anticodon. Each trna with a particular anticodon (ex. GAA) will always carry a specific amino acid (ex. Leucine)

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19 Translation Meanwhile, proteins and rrna have come together to build a ribosome. Ribosomes come in two sections called subunits. The two subunits sandwich themselves over a strand of mrna and a series of trnas. When this happens, translation is ready to begin Eukaryotic cells contain 100,000 s of ribosomes, all working simultaneously if necessary.

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21 1. Initiation Translation mrna strands are organized into codons, or combinations of three RNA nucleotides The first codon is always the same: AUG A trna with the anticodon that matches with this codon (UAC) then enters the ribosome and matches with the mrna, codon-to-anticodon

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23 2. Elongation Translation A second trna then enters the ribosome and matches anticodonto-codon. When it attaches to the mrna, the shape of the ribosome forces trna s to line up in a specific orientation. That orientation allows the amino acids that each trna are holding to break from their trna s and attach to each other.

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25 Translation The ribosome has three sites which are able to hold trna s. The A site, which is where trna s enter the ribosome and wait their turn. The P site, which is where the attachment of amino acids will occur (or, the formation of a peptide bond) The E site, which is where trna s will exit, leaving their amino acids behind.

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27 During elongation, only one amino acid is attached at a time. One trna must leave the ribosome before the next one can enter. The entire process to build and form a single protein and repeat takes around 15 minutes for eukaryotic cells. 3. Termination Translation When the ribosome reaches a stop codon (UAA, UAG, UGA) the mrna attaches to a release factor. The release factor breaks the final amino acid from the final trna, thus completing the protein chain.

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29 Translation Once the amino acid sequence is released from the ribosome, it undergoes folding and modification. The endoplasmic reticulum adds lipids, carbohydrates, or other proteins to the new protein The ER also handles the specific folding of the protein The golgi then wraps a vesicle around the protein for transport to it s destination. Meanwhile, the ER reattaches amino acids to the trna s that have just given their amino acids up. The mrna then is re-translated to build another protein, or is returned to the nucleus to be used for spare parts.

30 Codon Pattern Translation relies on the fact that 1) Every codon will match with a specific anticodon 2) Every trna with that specific anticodon will have a specific amino acid. Thus, each of the 20 amino acids in organisms must have at least one codon (some have up to six). As far as we know, the code is the same for every species on the planet

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35 Mutations The human genome contains 40,000 confirmed genes and an estimated 70,000 total genes to be found. The sequence of DNA to make these genes is around 3.3 billion nucleotides. Smallest human gene: organizational proteins called histones, used in cell division. 648 bp, no introns Largest human gene: Titin connectin, for muscle elasticity. 80,780 bp, 348 separate exons. Although ribosomes are able to correct mistakes in the moment, some do escape notice. There are two types of transcription/translation mutations you should be aware of.

36 Mutation #1: Point Mutations A point mutation is when only one nucleotide is incorrect. Even though it is only 1 nucleotide out of 3.3 billion, this one mistake has the potential to completely change an organism s health. Example: I have a pet cat. This could be I gave a pet cat; I wave a pet cat; I have a wet cat. I have a pet rat. As discussed, sickle cell anemia is a disease caused by a single point mutation that tells the cell to replace a glutamine with a valine

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39 Mutation#2: Frameshift Mutation Frameshift mutations are when at least one nucleotide is added or deleted from the DNA or RNA sequence The correct sequence of amino acids is dependent on maintaining the 3-nucleotide codon pattern. If one nucleotide is added or deleted, the codon pattern does not start at the right spot This results in an entire sequence of amino acids being incorrect.

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41 Extra Credit Questions (2 this time!) EACH question is worth an extra 5% on your essay exam You may check your answers with me ahead of time for a yes or no response as many times as you like. Question #1: Some genes can only be inherited from your mother, but males and females both receive these genes. How is this possible? Question #2: E. coli takes 40 minutes to fully replicate DNA. Under extreme circumstances, though, it can do a full cell division in only 30 minutes. How is this possible?