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The Molecular Structure of DNA FRANCIS HARRY COMPTON CRICK, JAMES DEWEY WATSON AND MAURICE HUGH FREDERICK WILKINS, 1962 ARTHUR KORNBERG AND SEVERO OCHOA, 1959 ROBERT W. HOLLEY, HAR GOBIND KHORANA AND MARSHALL W. NIRENBERG 1968 WERNER ARBER, DANIEL NATHAN AND HAMILTON O. SMITH, 1978 ANDREW Z. FIRE AND CRAIG C. MELLO, 2006 Francis Harry Compton Crick, James Dewey Watson and Maurice Hugh Frederick Wilkins, 1962, “for their discoveries concerning the molecular structure of nucleic acid and its significance for information transfer in living material” * DNA Deoxyribonucleic acid *More about Rosalind Franklin later The Laureates From left to right: Maurice Wilkins Max F. Perutz (Chemistry) Francis Crick John Steinbeck (Literature) James Watson John C. Kendrew (Chemistry) Not pictured here: Linus Pauling (Peace) Lev Landau (Physics) James D. Watson Francis Crick Maurice Wilkins Two important players who didn’t get the prize Rosalind Franklin Linus Pauling X-Ray Diffraction Three forms of DNA X-ray Diffraction of DNA DNA: The building blocks Keto-enol Tautomers The cross bridges are pairs made up of one purine and one pyrimidine 1953: Watson and Crick elucidate the structure of DNA http://www.dailymotion.com/video/xitlyu_life-storythe-race-for-the-double-helix-1-2_shortfilms DNA: basic structure Arthur Kornberg and Severo Ochoa, 1959, “for their discovery of the mechanism in the biological synthesis of ribonucleic acid and deoxyribonucleic acid” Kornberg (1918-2007): two major accomplishments (1956): • He isolated DNA polymerase I, the enzyme that links nucleotides (deoxyribose + base) through phosphate linkages • Using radioisotopes and two phosphodiesterase enzymes that cleave DNA only from the 3’ end, he showed that DNA is always synthesized from the 5’ to the 3’ end. Ochoa (1905-1993) initially studied intermediary metabolism. He was primarily interested in ATP and enzymes that catalyze oxidative phosphorylation. Working with Marianne GrunbergManago, he discovered what he thought was RNA polymerase. Grunberg-Manago felt that the enzyme was in fact a polynucleotide phosphorylase which breaks down RNA. She was right, but this was not shown until after the Nobel prize. Robert W. Holley, Har Gobin Khorana and Marshall W. Nirenberg, 1968 “for their interpretation of the genetic code and its function in protein synthesis” By 1959 it was accepted that DNA produced RNA which in turn somehow coded for proteins. What was not known was the length of each DNA codon (George Gamow theorized 3, as the minimal code for all 20 amino acids) or the actual code itself. Nirenberg and his post-doc Johann Matthaei used a cell-free E. coli extract with synthetic RNA and radioactive amino acids to deduce the code and the length of the codon*. • First they used repeat polymers such as UUUU… which produced PhePhePhe… • Next they used mixtures of known ratios of nucleotides to make the RNA: e.g., 3:1 U:G. • Thus P(UUU)= ¾* ¾ * ¾ , so 27 of every 64 triplets is UUU • P(UUG)= ¾ * ¾ * ¼, so 9 out of 64 triplets is two U’s and one G • P(UGG)= ¾ * ¼ * ¼, so 3 our of 64 triplets is one U and two G’s • Result: VAL, LEU and CYS are incorporated 1/3 as often as PHE, so their codes have two U’s and one G. But in what order? • Lastly they bound triplets to ribosomes and added the necessary ingredients. They found the specific Aas that bound to the triplet-loaded ribosomes • In the end, they identified 47 of the 64 possible triplets. *Ochoa was trying much the same thing, in a much better-equipped lab. Since Nirenberg and Matthaei were working at NIH, many NIH scientists pitched in to help get a Nobel for the NIH. Holley, Khorana and Nirenberg, 1968 Khorana, working independently, used a different approach: he synthesized DNA with only a few bases in sequence, e.g., ATGATG…. Using this method Korana produced mRNAs of defined sequence and thus resolved some ambiguous codes. For example, mRNA GUGUGUGU---, has two possible codons, GUG or UGU. It produces CysValCysVal…Knowing that UGU codes for Cys, GUG must code for Val. Holley, an organic chemist, was able to determine the structure of transfer RNA, the RNA which at one end attaches to a specific amino acid and at the other has a code complementary to that on the messenger RNA. Transfer RNA and the Genetic Code Werner Arber, Daniel Nathans and Hamilton O. Smith, 1978 “ for the discovery of restriction enzymes and their application to problems of molecular genetics” Arber discovered these enzymes in the early 1960s when he analyzed an apparently obscure phenomenon in bacteria, discovered 10 years earlier by Bertani and Weigle, called host-controlled modification. Arber showed that this phenomenon was caused by a change in DNA and apparently served to degrade Foreign DNA. Arber postulated that bacteria contain enzymes with the capacity to recognize and bind to precise sequences in the DNA and sever it at those loci. Smith verified Arber's hypothesis. He purified one restriction enzyme and showed that it could cleave foreign DNA. He determined the chemical structure of the regions of DNA which were severed by the enzyme and discovered certain rules which later could be applied to other restriction enzymes. Nathans pioneered the application of restriction enzymes in genetics. He constructed the first genetic map using restriction enzymes by cleaving the DNA from a monkey virus. The methodology devised by him for this purpose was later used by others to construct increasingly more complicated maps. Plasmids and Restriction Enzymes Andrew Z. Fire and Craig C. Mello, 2006, “for their discovery of RNA interference-gene silencing by double-stranded RNA” Fire and Mello in 1998* found that if they injected fragments of double-stranded RNA (dsRNA) into C. elegans, they could selectively turn off certain genes if one strand of the dsRNA was complementary to the gene on the DNA. We now know that such exogenous dsRNA, or RNAi, uses an ancient mechanism for regulating gene expression using micro RNA (miRNA) produced by the cell itself. Essentially, RNAi mimics miRNA. miRNA is produced in the nucleus. After some processing there it moves into the cytoplasm. There it is cleaved by an enzyme called Dicer, and is incorporated into a complex called RISC (RNA-Induced Silencing Complex), which binds to messenger RNA (mRNA) and modulates expression of that mRNA into proteins. Introduced dsRNA differs from miRNA in that it forms a RISC which is much more specific for the mRNA sequence of its target. *Fire, et al., “ Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans” Nature 391, 806, 1998) Interference RNA Why natural RNAi? Two good reasons for this defense mechanism: • Viruses • Some viruses are dsRNA viruses (e.g., rotavirus which causes gastroenteritis in children, and picobirnaviruses that cause diarrhea). • This dsRNA could trigger the RNAi system and lead to the suppression of virally-induced mRNA • Jumping genes • Many transposons are delimited by terminal inverted repeats, which could potentially give rise to dsRNA which in turn could trigger the RNAi system. Viral mRNA Animation of RNAi Mechanism Therapeutic and Research uses of RNAi Therapy: Dominant Negative Mutations: One allele produces an abnormal protein which outcompetes the normal protein for the appropriate receptor. Many of these mutations are SNPs. Drugs cannot differentiate between the normal and mutant protein, but RNAi might be able to target and degrade the mutant mRNA Viral Diseases: Introduce a dsRNA (or an artificial DNA that transcribes into a dsRNA (hairpin) which is complementary for the viral mRNA. Cancer: Currently clinical trials are underway for among other things hepatitis C and HIV. RNAi could be used to knock out cancer-producing genes, or perhaps even to upregulate cancer-suppressing ones. Research: Learn the function of a gene by blocking its expression. Delivery of RNAi In any therapeutic use of RNAi the difficulty is to get the RNAi to its target cells. This includes preservation in the bloodstream, prevention of immune response, and entry into the specific target cell. The CRISPR-CAS 9 System: Next Nobel Prize?? Fig. 1 Overview of the four CRISPR/cas systems present in Streptococcus thermophilus DGCC7710. Fig. 2 Overview of the CRISPR/Cas mechanism of action. Philippe Horvath, and Rodolphe Barrangou Science 2010;327:167-170 Published by AAAS Fig. 3 CRISPR interference. Philippe Horvath, and Rodolphe Barrangou Science 2010;327:167-170 Published by AAAS DNA surgeon. With just a guide RNA and a protein called Cas9, researchers first showed that the CRISPR system can home in on and cut specific DNA, knocking out a gene or enabling part of it to be replaced by substitute DNA. More recently, Cas9 modifications have made possible the repression (lower left) or activation (lower right) of specific genes. Published by AAAS Elizabeth Pennisi Science 2013;341:833-836 Editas Medicine, Inc.