Molecular Genetics. 1. To understand the structure of DNA. 2. To understand how the genetic code is transcribed and translated into protein.

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Anabel Hines
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1 Molecular Genetics Objectives: 1. To understand the structure of DNA. 2. To understand how the genetic code is transcribed and translated into protein. Each gene has the instructions to make a single polypeptide chain. These instructions are part of the genetic code. Polypeptide chains are the structural units of proteins. A polypeptide chain is made of many amino acids bonded together. The key to the genetic code is the sequence of nitrogenous bases along one side of the DNA molecule. To construct a protein you must know the order of the bases. The code is written in three letter words. Each of these codons (or triplets) tells the cell which amino acid should come next when building a protein. When a specific protein is required by the body, regions of the double helix unwind, so that a cell gains access to the genes that contain the coded information to make that protein. Protein synthesis has two steps: transcription (copying the message from DNA into RNA that can be moved to the cytoplasm) takes place in the nucleus and translation (translating it into protein) occurs in the cytoplasm. Both steps require molecules of RNA (ribonucleic acid). I. The Basics of DNA Structure DNA molecules are composed of small building blocks called nucleotides: A. Each DNA nucleotide is composed of three smaller molecules bonded together: one nitrogenous base, one phosphate and one five-carbon sugar (deoxyribose). B. Four different types of nucleotides are needed to build a DNA molecule. Each of these four nucleotides has a different nitrogenous base: adenine, cytosine, guanine or thymine. C. DNA consists of a structure analogous to a twisted ladder: The two sides of the ladder are made of alternating sugar and phosphate molecules. D. Each sugar molecule is attached to one nitrogenous base. The two strands of DNA are attached by hydrogen bonds between the nitrogenous bases on each side of the ladder. Each nucleotide base only bonds with one specific partner. The combination of two bases is called a base pair. Adenine ALWAYS pairs with Thymine (A-T). 1

2 Guanine ALWAYS bonds with Cytosine (G-C). Building a Model of DNA A. Work in pairs. Get a DNA model kit. B. Push the support rod firmly into the gray stand C. Separate the parts of the DNA model according to the description in Table 1 (below), verifying that you have the correct number of parts. D. Match pairs of bases then slide them onto the pole, placing clear spacers between each pair. Be sure that, at this stage, all the outside holes in the bases line up vertically. E. Build two backbones of ribose and phosphate subunits. First, push the lower corner (not peak) peg of the deoxiribose unit into the hole on the phosphate and orienting the phosphate point down. Then push the next ribose subunit onto the peg on the phosphate. When you have two equal-length backbones, attach them to the bases. F. Starting at the bottom of the model, connect each deoxiribose to the bases on the pole (using the deoxiribose peak peg), allowing the backbone to spiral around the pole. Connect the second backbone in reverse of the first (i.e. one starts with a phosphate group and the other starts with a ribose group). If you cannot connect the second backbone, then some of your bases are backwards see point D. Table 1. Parts of the DNA model. Parts of the DNA molecule Description of Model Part (number) Deoxyribose red pentagon (24) Phosphate purple triangle (24) Adenine (A) base blue (6) Thymine (T) base orange (6) Guanine (G) base green (6) Cytosine (C) base yellow (6) Questions: 1. How many hydrogen bonds connect each pair of nucleotides? 2. Do the backbones run in the same direction (parallel)? 3. Assume that the model you just built is an exact representation of your DNA code. Would you use the same bases to construct your lab partner s DNA? 4. Would you assemble the bases in the same order to make a model of your lab partner s DNA? 5. What is your nucleotide sequence? 2

3 II. DNA to RNA: During transcription, DNA bases of a gene are copied (transcribed) to a single strand of RNA, called messenger RNA (mrna). As with the DNA, mrna is divided into coded three letter words called codons. The base pairing rule is used to form messenger RNA with one exception: RNA have uracil (U) in place of thymine (T). Modeling transcription A. Get a RNA model kit. B. Separate the parts of the RNA model according to the description in Table 2 (below), verifying that you have the correct number of parts. C. Slip your DNA model off of the central pole and gently separate the two strands. Choose one strand, and build a complementary RNA by pairing RNA bases to the DNA bases and then hooking sequential bases together with intervening ribose and phosphate subunits. Be sure that the RNA bases are connected to the peak peg on the ribose and the phosphate to the bottom peg. D. Note that if you have long runs of the same base pairs in your DNA model, you may not be able to transcribe the entire gene. Table 2. Parts of the RNA model. Parts of the DNA molecule Description of Model Part (number) Ribose dark red pentagon (12) Phosphate purple triangle (12) Adenine (A) base blue (3) uracil (U) base light blue (3) Guanine (G) base green (3) Cytosine (C) base yellow (3) Transfer RNA (trna) dark red, 2 Amino acids pink, light green (1 each) Questions: 1. What is the sequence of the mrna strand? 2. Compare it to your DNA sequence (question #5 above). Does it compliment the DNA? If not, can you explain why? 3. How many triplets are present in your RNA model? 3

4 4. How many amino acids will be present in the protein made from your model? III. RNA to protein 1. A cell needs amino acids to construct proteins. The amino acids are carried to the ribosomes by another type of RNA molecule, called transfer RNA (trna). A trna has two functional ends. One end picks up amino acids in the cytoplasm. 2. The other end is called the anticodon. It contains three nitrogenous bases that can form a base pair with a matching codon in the mrna. 3. Each type of trna can carry only one type of amino acid. There are enough different types of trna molecules to carry all the different types of amino acids needed to make your body s proteins. 4. Where do the trna molecules take the amino acids? They take them to ribosomes, organelles in the cytoplasm where proteins are manufactured. Ribosomes are made of proteins plus a third type of RNA called ribosomal RNA. 5. Ribosomes read messenger RNA codons and accept amino acids brought by trna molecules. Ribosomes help bond amino acids together in the order specified by the messenger RNA codons to construct the polypeptide chain. Modeling Protein Synthesis 1. Gently separate the RNA from the DNA. Set the DNA strand aside. 2. Dismantle one end, leaving the model with six nucleotide bases. 3. Using one of the trna molecules, construct a complementary sequence to the first triplet in the RNA. Repeat with the second trna and second triplet. The trna triplets are anticodons to the codons in the mrna. 4. Attach an amino acid to the top peg on each trna. 5. Bind the first trna to the first triplet, and the second to the next. The two amino acids should be close enough to hook together. IV. Building a Real Protein (Practicing transcription and translation) 4

5 Imagine the following situation: you are about to give birth (this may be tougher for some of us than others). The brain produces the hormone oxytocin (a small protein), which causes uterine muscles to contract for childbirth. Following birth, this same hormone causes muscles in the mammary glands to contract, releasing milk to nurse the baby. You have to admit, that s QUITE a little protein! Indeed, in the last few years it has been discovered that this same protein is involved in establishing not only bonding between mother and child, but also bonding between parents and among groups of close friends. So using the steps of Protein Synthesis, let s build the protein Oxytocin. We re using Oxytocin since it is one of the smallest proteins with only nine amino acids in the polypeptide chain. 1. Transcription (nuclear sequence to mrna) Transcribe the DNA sequence for oxytocin below (this is the entire sequence it is a very small protein!) Remember that in RNA, thymine gets replaced with uracil, and that amino acids are grouped in threes (i.e. DNA triplets). DNA triplets mrna codons amino acids ACG ATG TAT GTT TTG ACG GGA GAC CCC trna anticodons 2. Translation Each amino acid is carried by a specific trna molecule to the ribosome /mrna complex. Table 3 presents the genetic code. Use this to determine the sequence of amino acids in this portion of the oxytocin molecule. Determine the anticodon that each trna would need to match the mrna codon and add that to the table above. Questions: What kind of functional group does the second amino acid in this protein have? (Use figure at end of this document). What do you think would happen if this amino acid was replaced by phenylalanine? Glycine? Explain your thinking. 5

Interestingly, searching for variation in the oxytocin gene has thus far been futile: variants are known for the DNA regions around this gene, but

6 Interestingly, searching for variation in the oxytocin gene has thus far been futile: variants are known for the DNA regions around this gene, but no variants have been found for the gene itself. Every human tested produces this exact oxytocin protein. Speculate on why this might be the case. 6

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Transcription and Translation of DNA

Transcription and Translation of DNA Genotype our genetic constitution ( makeup) is determined (controlled) by the sequence of bases in its genes Phenotype determined by the proteins synthesised when genes