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I. DNA History of the Role of DNA Since the late 1800’s, scientists knew that information was passed from parent to offspring in a predictable way. As biology and genetics advanced, scientists were able to begin narrowing down exactly which substance in the cell was responsible for containing the genetic information. From 1910’s to the 1940’s, there was a debate between whether nucleic acids or proteins were the source of genetic information. And then a few key experiments in the first half of the 1900’s shed some light on the subject. A. 1928: Frederick Griffith – Bacterial Transformation 1. Griffith was trying to figure out how bacteria make people sick and was studying pneumonia. a. Had isolated two different strains of bacteria (same species, different variety) in mice b. S strain – caused pneumonia in mice, colonies have smooth edges c. R strain – did not cause disease in mice, colonies have rough edges 2. Griffith’s Experiments a. The following experiments were done as controls: i. Living S bacteria injected into mouse: mouse develops pneumonia and dies ii. Living R bacteria injected into mouse: Mouse stays healthy iii. Heat killed S bacteria injected into mouse: Mouse stays healthy b. But when Griffith injected heat-killed S bacteria mixed with live R bacteria into a mouse, the mouse developed pneumonia and died! By themselves, neither type of bacteria should have made the mouse sick. i. When the dead mice were examined, Griffith found that the lungs were full of S bacteria. ii. How was this possible if the S bacteria were dead when he injected them into the mouse? Griffith’s Experiments - Transformation Hereditary material from the heat-killed S cells transforms R cells. The transformed R cells kill the mouse. c. Somehow, the heat killed S bacteria passed their disease causing ability to the harmless R bacteria. Griffith concluded that some chemical factor transferred from the heat killed S bacteria to the live R bacteria. He reasoned that the chemical factor must contain information that could change the harmless bacteria into the disease causing bacteria. d. Griffith also found that the newly disease causing bacteria passed down the characteristic to their descendents, so he figured the chemical factor must be a gene. e. Griffith called this phenomenon transformation. f. Transformation: a change in the genes or traits of an organism due to the addition of outside DNA by a cell B. 1944: Oswald Avery – Cause of Transformation 1. Avery and his team wanted to determine which molecule in the heat killed bacteria was most important for transformation to occur. If they were successful, they might help to reveal the chemical nature of the gene. 2. The experiments: a. Avery extracted a mixture of various molecules from the heat killed bacteria, most notably protein, lipids, carbohydrates, RNA, and DNA. b. Enzymes were used to separately destroy each of the molecules in heat killed bacteria. So one batch of heat killed bacteria had proteins destroyed, another had the RNA destroyed, and so on. c. It was found that after proteins, lipids, carbohydrates, and RNA were destroyed, transformation still occurred. It was only after the DNA was destroyed that transformation did not happen. Avery concluded that DNA must be the molecule responsible for transformation. 3. It would take further experimentation for the scientific community to accept that DNA was definitely the material of the gene. Some scientists still felt that protein was more suited to the function of the gene. The work of Griffith and Avery are the foundation on which further work would build to show that the gene was indeed composed of DNA. C. 1952: Alfred Hershey and Martha Chase – Protein or DNA? 1. Hershey and Chase studied a bacteriophage to find out if it was indeed DNA or protein that was the material of the gene. 2. Bacteriophage: a virus that infects bacteria a. At the time, biologists knew that most viruses were composed almost entirely of DNA and protein b. They also knew that the virus would quickly turn the bacterial cell into a factory for making more viruses. c. But they didn’t know how the virus reprogrammed the cell to produce viruses. Was it the DNA or the protein that was responsible? Which one actually entered the bacteria? 3. The Experiment a. Viruses were grown in cultures containing radioactive isotopes. Since they are radioactive, the isotopes could be easily detected and would act as “tags” for the protein and for the DNA. b. One culture was grown in radioactive phosphorus and another in radioactive sulfur. Why these two elements? Proteins contain almost no phosphorus and DNA contains no sulfur. c. So the virus’s protein coat was marked with radioactive sulfur and the virus DNA was marked with radioactive phosphorus. d. The marked viruses were then mixed with bacterial cells, given time for the viruses to infect the bacteria, and then separated the viruses from the bacteria. e. Hershey and Chase then checked the bacteria for radioactivity. They found radioactive phosphorus, the marker on the viral DNA. It was the DNA that was injected into the bacteria, not the protein. DNA was the genetic material, not protein. Alfred Hershey and Martha Chase used different radioactive markers to label the DNA and proteins of bacteriophages. The bacteriophages injected only the DNA into the bacteria, not proteins. From these results, Hershey and Chase concluded that the genetic material of the bacteriophage was DNA. 4. Hershey and Chase confirmed Avery’s results. Their work was crucial in convincing the scientific community that DNA was the genetic material found in cells. II. Structure of DNA While Hershey and Chase worked to confirm Avery’s results, the race was on to find the structure of the gene, or DNA. We’ll be going into more detail of the key people involved later, but we’re going to look into the actual structure now. Structure of DNA Remember: genes control certain traits, genes are sections of DNA I. Structure of DNA (deoxyribonucleic acid) A. Made of nucleotides 1. nucleotides have 3 main parts a. sugar (deoxyribose) S b. phosphate group P c. nitrogenous base (see below) 2. 4 different nitrogenous bases can be used in a nucleotide a. adenine (A) b. guanine (G) c. cytosine (C) d. thymine (T) 3. DNA formed by 2 strands of nucleotides linked together a. “double helix shape” twisted ladder b. sides of the ladder are alternating molecules of sugar (deoxyribose) and phosphate c. “rungs” of ladder are 2 bases bonded together by hydrogen bonds: i. Adenine always bonds with Thymine – held by 2 hydrogen bonds ii. Cytosine always bonds with Guanine – held by 3 hydrogen bonds iii. A = T and G = C is known as Chargaff’s Rule(s) – based on his experiments in 1950 showing that in samples of DNA, amounts of adenine and thymine are always equal, and amounts of guanine and cytosine are always equal 4. James Watson and Francis Crick – published their work showing the structure of DNA in 1953 (more detail later) Names and Dates: Supplement to DNA Notes Watson and Crick work together at the Cavendish Lab at Cambridge University in England. Sir Lawrence Bragg was in charge at the Cavendish. Watson and Crick take the approach of model building to solve the DNA puzzle. February 28, 1953: Crick announces in the English pub “The Eagle” that he has found “the secret of life.” Watson and Crick published their work later in 1953. Two other articles are published with their work, one of which is Rosalind Franklin’s article. Her article is modified to make it appear that her work was based on and supports Watson and Crick’s conclusions about the structure of DNA. In reality, it’s the reverse – Watson and Crick’s work is based on Franklin’s work that they deviously obtained through Maurice Wilkins, Franklin’s colleague. James Watson, Francis Crick and Maurice Wilkins received the Nobel prize in 1962 for their work on DNA structure and how the DNA molecule can function to carry genetic information. Rosalind Franklin: 1. She worked at King’s College in London, England. The head of her lab was J. T. Randall. Maurice Wilkins was a colleague of hers at King’s. She and Wilkins often had personality clashes, partly because Randall never clarified each one’s role at the lab. 2. She performed research on the DNA molecule using X-ray crystallography to take pictures (she was one of the best crystallographers in the world); this research was the basis of the double helix shape to DNA that Watson and Crick are so famous for discovering. Franklin took the approach of collecting data and then analyzing the data for finding the structure of DNA. The idea of a helical shape for DNA was all Rosalind’s. Without her work, Watson and Crick would probably have not figured out the DNA molecule before anyone else. 3. She died at age 37 in 1958, before Watson publishes his book and before the Nobel Prize is awarded. 4. She was never acknowledged for her contribution to the structure of DNA until Watson described her as a horrible person in his book “Double Helix” (published in 1968). Everyone who was familiar with the DNA story objected to Watson’s portrayal of her and the fact of Rosalind’s work being so important was brought to the public’s attention. 5. Beyond DNA research, Franklin contributed vast amounts of knowledge to science in the areas of coal, viruses and their structure and function, and even helped to create a far better gas mask for war torn Britain during WWII. Her knowledge of X-ray diffraction would lead to her travels throughout the US to give lectures at many of the most renowned universities. Maurice Wilkins: Was the “assistant director” of the lab that Franklin worked in; he was good friends with Crick and was most likely the conduit through which Franklin’s work (including Photo 51) is given to Watson and Crick; received the Nobel Prize with Watson and Crick in 1962. Alfred Nobel: Was a major business man in mid to late 1800’s; invented dynamite and made his huge fortune from it; his brother died and a newspaper printed Alfred’s obituary by accident and called him the “merchant of death” (dynamite killed a lot of people in demolition accidents); Alfred hated the thought of his legacy being such a terrible one so when he died he left most of his money for the establishment of the Nobel Prizes; it’s the highest award a person can receive and covers lots of different categories (literature, physics, medicine, peace...) Erwin Chargaff: 1. He studied DNA and analyzed how much thymine, cytosine, adenine and guanine were in each sample. He found that the amounts of thymine and adenine were always equal, and the amounts for cytosine and guanine were always equal. 2. The result was Chargaff’s Rule: A = T and C = G DNA Replication I. Replication – process of copying DNA A. The structure of DNA lends itself well to being copied. 1. Need to be able to copy DNA for cell division and for reproduction 2. The accuracy of replication is impressive – only about 1 error in every 10 billion nucleotides – and is especially remarkable considering the speed of the process. However, despite proteins that check for damage in the DNA or mistakes in the copying process, replication is not 100% foolproof. And these mistakes, though rare, means that genes are sometimes altered during replication. 3. The rules of base pairing (A-T, C-G) factor heavily into the process 4. Remember, replication occurs during the later part of interphase. B. Prokaryotes vs. Eukaryotes Prokaryotes: 1. In most prokaryotes, there is a lot less DNA to copy. Most have a single circular DNA molecule, its single chromosome, located in the cytoplasm. 2. Replication begins at a single point in the chromosome and proceeds in two directions until the entire chromosome is copied. Eukaryotes: 1. Eukaryotic chromosomes are generally much bigger than those of prokaryotes. 2. Replication in eukaryotes may begin at hundreds or even thousands of places on the DNA molecule, each called an origin of replication (special sites of DNA with specific sequences of nucleotides). At each origin, replication will proceed in two directions. 3. As replication proceeds, these areas eventually will meet up with each other. Multiple areas being copied at once makes the process much faster than having a single origin of replication. C. How Replication Happens 1. The two strands of the double helix separate, or unzip. This is done by a series of enzymes. Remember that the base pairs (the rungs of DNA) are held together by hydrogen bonds. These are relatively weak compared to the covalent bonds holding the sugars and phosphates together (sides of the ladder of DNA). 2. This unzipping allows two replication forks (areas where the DNA is unzipped and new DNA is forming) to form. Each side or strand of the unzipped DNA will be a template for a new strand to form. 3. New nucleotides are added to each side according to the rules of base pairing (A with T, C with G). These nucleotides are called “free nucleotides” and are free floating in the nucleus. The enzyme called DNA polymerase joins individual nucleotides to the unzipped sides to form a new strand (or side) of DNA, completing the helix. DNA polymerase also “proofreads” the new strand of DNA to avoid errors. 4. The sugars and phosphates on the sides covalently bond together to form a new backbone. D. Semiconservative Process 1. At the end of replication, there are two copies of the DNA molecule. 2. In each DNA molecule, one strand (or half of the molecule) is from the original DNA that we started with. The other strand was made from free nucleotides and is the “new” strand. 3. This model of copying, where the “copy” actually contains half of the original molecule, is called the semiconservative model. Containing an original strand helps to prevent mistakes. DNA Replication I. The Role of RNA A. Despite finding the structure of DNA, it was not at all clear how a gene actually works. To find this answer, a lot more research and experimentation had to be done. 1. Part of the answer was provided when researchers found that another nucleic acid, ribonucleic acid or RNA, was involved in putting the genetic code into action. 2. In decoding the information of DNA, part of the base sequence of DNA is copied into a section of RNA. 3. The RNA then has the instructions to direct the next step, which is making proteins. Proteins help to determine the characteristics of an organism and are a major part of genes being expressed. B. RNA and DNA 1. Both DNA and RNA are nucleic acids, but there are several structural differences, summarized in the chart below. DNA Double stranded RNA Single stranded Base pairs: A–T C–G Base pairs: C–G A–U U=uracil; replaces thymine Deoxyribose is the sugar Ribose is the sugar 2. Comparing the roles of DNA to RNA: DNA is like the complete set of blueprints to build a shopping mall. RNA is a disposable copy of one tiny section of those blueprints, like the plumbing for the Starbucks inside the mall. As a builder, you would never let someone borrow your blueprints. They are too valuable. Similarly, the DNA doesn’t leave the nucleus. Instead, RNA is used to pass along the information. II. Types and Functions of RNA A. There are many types of RNA, but the majority of them are involved with one job – to make proteins. 1. RNA controls the assembly of amino acids into proteins. 2. Recall that proteins are made from chains of amino acids, like beads in a necklace. A single chain of amino acids is a polypeptide. Proteins can be made of a single polypeptide or many polypeptide chains bonded together. 3. The properties of a protein are determined by the order in which different amino acids are joined together. (The order of the amino acids is determined by the order of the nucleotides in the DNA, but you’ll see that later. ) 4. Recall as well that proteins are made in the ribosomes of the cell. B. While there are other types, we will discuss the 3 main types of RNA used in protein synthesis (which is the process of making proteins). 1. Ribosomal RNA (rRNA) – ribosomes are made of 2 subunits and each subunit is composed of several rRNA molecules and lots of different proteins 2. Messenger RNA (mRNA) – used to send information from the DNA to the ribosome; serves as a disposable copy of the information in the DNA 3. Transfer RNA (tRNA) – each tRNA molecule has an amino acid attached to it; the tRNA brings the amino acid to the ribosome – when the tRNA and the mRNA match up, the amino acid is added to the chain of amino acids III. Protein Synthesis A. Protein synthesis : process of making proteins 1. Most genes contain instructions for assembling amino acids into proteins. 2. Proteins play a large role in determining an organism’s characteristics. 3. RNA is integral to this process. 4. There are two parts to protein synthesis – transcription and translation B. Transcription 1. During transcription, a segment of DNA is copied into a complementary strand of mRNA. a. Requires the enzyme RNA Polymerase, which binds to the DNA and separates the strands. b. RNA polymerase then uses one strand of DNA as a template for the nucleotides being assembled into a strand of mRNA. 2. How does the RNA polymerase know where to start on the DNA? a. The enzyme only binds to promoters. Promoters are regions of the DNA that have specific base sequences that signal RNA polymerase exactly where to start making mRNA. b. Other signals result in transcription stopping. 3. Transcription - Prokaryotes vs. Eukaryotes a. In prokaryotes, the mRNA (made in the cytoplasm) goes straight to the ribosome for the next step in protein synthesis – translation. b. In eukaryotes, the mRNA (made in the nucleus) is first spliced inside the nucleus. i. Splicing: removing the parts of mRNA that aren’t needed ii. Introns are the segments of mRNA that are removed. Exons are the segments that are spliced back together. (exon expressed) iii. We’re not entirely sure of the reasons behind splicing. 4. After splicing, the mRNA leaves the nucleus and goes to the ribosome for the second part of protein synthesis, translation. Splicing is pictured above. Splicing only occurs in eukaryotes. Transcription is pictured above. Remember that in prokaryotes, transcription happens in the cytoplasm. In eukaryotes, it occurs in the nucleus. Note that the mRNA is single stranded & has uracil, while DNA is double stranded & has thymine. C. Translation 1. Translation – converting the information in the mRNA into a chain of amino acids (polypeptide or protein) 2. How can a code of just 4 letters carry instructions for 20 different amino acids? a. The genetic code (in the form of mRNA) is read three “letters” at a time, so that each “word” is 3 bases long and corresponds to a single amino acid. b. Each three letter “word” in mRNA is known as a codon. i. Codon: a 3 base section of mRNA ii. Each codon specifies a single amino acid that is to be added to the polypeptide chain c. How do you “read” the codons to find the right amino acid? USE THE CHART!!! d. Since there are 64 different combinations of the 4 letters (43), most amino acids have more than one codon that will code for it. For example, how many codons are there for the amino acid called Leucine? e. There are also start and stop codons, which act as punctuation marks of a sort. i. The start codon (AUG) is for Methionine. ii. How many “stop” codons are there? 3. Steps of Translation a. Begins when a ribosome attaches to an mRNA molecule in the cytoplasm. b. The mRNA is read one codon, or 3 bases, at a time. c. As each codon passes through the ribosome, tRNA’s bring the proper amino acids to the ribosome. i. Each tRNA molecule carries just one kind of amino acid. ii. Each tRNA has 3 unpaired bases sticking out. These 3 bases on the tRNA are called the anticodon. iii. Each tRNA is complementary to (bonds with) one mRNA codon. iv. Only when the anticodon and the codon bond together is the amino acid accepted and added to the chain. d. The ribosome helps a peptide bond to form between the amino acids as they are added. e. As each amino acid is removed from the tRNA, the now “empty” tRNA’s are released from the ribosome. They will “recharge” with another of the same amino acid in the cytoplasm to be used again. TRANSLATION B. The Polypeptide “Assembly Line” The ribosome joins the two amino acids – Methionine and phenylalanine – and breaks the bond between methionine and its tRNA. The tRNA floats away from the ribosome, allowing the ribosome to bind another tRNA. The ribosome moves along the mRNA, binding new tRNA molecules and amino acids. Remember: DNA RNA Protein!!!!