weber uiuc edu 21 September 2007 MCB150 Lecture 12 Lecture 12 21 September 2007 Announcements More broken hand notes Exam I key posted on web site They reserve the right to lower the scale at the end of the semester if it is deemed necessary o They have not had to adjust the scale yet o The points we need are already identified o The scale will never be raised no curve up either Direct questions about grading to Alejandra Stenger in 252 Davenport Questions about problems are to be handled via personal visits only no email no posting on WebCrossing Honors concentration need to meet to understand requirements We are now in Unit 2 Material The Central Dogma DNA RNA Protein This is what we are going to spend the next month learning about this DNA content hereditary content gives the cell blueprints for making molecules Yet the information in the DNA is not directly used to make a protein Instead it goes through an intermediate RNA o Synthesized from a DNA template called TRANSCRIPTION o In prokaryotes it happens in the cytoplasm o In eukaryotes it happens in the nucleus Use the RNA to create a protein via a process called TRANSLATION DNA also has enough information to faithfully replicate the entire genome This flow of information is called the Central Dogma of molecular biology Discovering the Function of DNA The history of DNA Today for the names that we are covering we need to know their role 1940 s hereditary information is found to be on chromosomes o Chromosomes were know to be made from chromatin Chromatin contains a DNA component and protein component o We knew proteins have 20 different Amino Acids o We knew DNA was made up of four different nucleotides When considering the variety of proteins out there realize it is a huge number o If there were 20 letters in amino acids but only 4 in nucleotides which would be the logical hereditary choice o They thought amino acids were used as the genetic material o It seemed logical that the protein was the carrier of the hereditary information o Read the book on the experiments o It was eventually learned that DNA was the one that carried the info Thus the 3D structure of DNA was now the biggest challenge The structure would give info on how to pass information from DNA to proteins Chargaff s Rules 1949 Chargaff was interested in the biochemistry of nucleotides Page 1 of 4 weber uiuc edu 21 September 2007 MCB150 Lecture 12 The conclusions he came to are today known as Chargaff s Rules When comparing adenine in different species there are different concentrations across the species BUT The interesting thing within experimental error the percentage of adenine equaled the percentage of thymine and the percentage of guanine equaled the percentage of cytosine o The ratio of A T is 1 1 while G C is 1 1 o He didn t know about DNA about base pairing etc These findings sat unappreciated for a number of years Rosalind Franklin Maurice Wilkins John Randall was PI of lab Had Maurice Wilkins an expert in X ray diffraction studies o He could prepare samples really well but didn t know how to X ray crystallography Rosalind Franklin was an expert in the field o She was not a subordinate to Wilkins o She was not a people person o Randall led them on They looked at the scatter pattern from the x rays Their conclusions included that it is a helical molecule with uniform width Franklin also concluded that the sugars phosphates were on the outside with bases on the inside James Watson American Francis Crick British Worked closely with Wilkins They wanted to beat Linus Pauling Franklin didn t want to release the information o Wilkins shared the data anyway Watson and Crick didn t do the research but just built the model They used Wilkins Franklin s data to make the model They realized that the DNA was actually a double helix o It explained Chargaff s Rules and the x ray results that Franklin found o It puts the sugars and phosphates on the outside the bases on the inside Published a one page article in Nature saying here is the structure 1962 Nobel was given to Watson Crick and Wilkins o Franklin had died in 1958 o She died of cancer Didn t realize how bad X rays were and it killed her DNA Chains How are they held together We know that covalent phosphodiester bridges hold the single strand together They figured it was hydrogen bonding o This the only way that they fit together and maintain a stable exact width o Purines go opposite pyrimidines o Will not get two pyrimidines or two purines opposite one another The groves in the DNA o There is a major and minor grove in DNA o This is useful to get in there to access the information Page 2 of 4 weber uiuc edu 21 September 2007 MCB150 Lecture 12 o Can peel apart the regions through the major groove Pairing of purines and pyrimidines only one way that they fit o Adenine will only fit opposite Thymine o Guanine will only fit opposite Cytosine These are called complementary or Watson Crick base pairs DNA Strands are Antiparallel One has 5 at top 3 bottom The other is 3 at top 5 bottom The unoccupied phosphate is the 5 end The unoccupied hydroxyl is the 3 end The run in parallel but face in different directions antiparallel We now see the structure that all the data points to DNA Denaturation What can we do to DNA to learn about its properties Separate the strands of DNA called denaturing o Remove stabilizing forces to get the strands separate o This does not involve removing nucleotides Stabilizing forces o Hydrogen bonds between base pairs o The nitrogenous bases are all stacked in the middle because they are also hydrophobic o Stabilization is helped by hydrophobic base stacking interactions We can use heat acids bases urea formamide etc o Heat is probably most commonly used denaturant o Don t be so severe to break off nucleotides o Anything that breaks about the hydrogen bonds The arrow goes in both directions o DNA will refold on its own o When you denature the strands will find themselves again Complementary bases will anneal Denatured DNA will anneal renature o It does not matter where the complementary pair comes from The essence of making new DNA is that the DNA does not care where the bases come from as long as they are complementary o Annealing will happen as soon as denaturation is relieved Why Unwind DNA Most DNA techniques require ssDNA Single strands will come back together into dsDNA any time complementary bases are together o With RNA uracil replaces thymine o Uracil will hydrogen base pair with