MCB 502A-2014. Lecture #2DNA Structure— The quest for structure of DNA— Discussion of the DNA structure— The basic configurations of the natural DNA molecules. The chromosome.— The four DNA basesDNA denaturation and renaturation / annealing— Denaturation of nucleic acids, by temperature— Denaturation of nucleic acids — low osmolarity, pH, polar solvents— Renaturation— Instantly-reassociating DNA— Hybridization reveals evolutionary relationship— Hybridization is used to detect and quantify DNA and RNAThe quest for DNAstructure-1What researchers new about the DNA structure before 1950:1. In the 40s, it was realized that DNA could not be atetranucleotide, because its molecular weight is huge andvaries. DNA was imagined as long linear chains of fournucleotide repeats.2. Preliminary crystallographic characterization of DNA,done by Bill Astbury in 1938, concluded that DNA materialis very densely packed. To explain this DNA density,Astbury proposed that the DNA structure is a single chainwith base and sugar pairs in the same plane and stacking.Besides introducing the biologically-critical concept of DNAbase stacking, Astbury also correctly determined thedistance between neighboring nucleotides at 3.4Å.3. The mysterious Chargaff rule (A=T, G=C) the nature ofwhich nobody, even Chargaff himself, understood.William Astbury0.34 nm"pile of pennies"sugar baseBy the way…Bill Astburry was the first to use the term "MolecularBiology" his his 1950 Harvey Lecture.— Scientists at that time objected, of course, arguingthat "Ultrastructural Biology" should be quiteadequate to describe "three-dimensional andstructural biology at the atomic level".— In Europe of that time, laboratories had their ownnames, decided at the University level. Astburywanted his lab at the Universiyty of Leeds to becalled the Laboratory of Molecular Biology.— However, the University handled down a differentname: "the Department of Biomolecular Structure".One of the arguments from the committee was: "hemay know something about molecules, but he knowsprecious little about Biology…"— Astbury was unphased and proposed the term"Molecular Genetics" in 1961!Astbury at his Department of TextilePhysics at the University of Leeds.The quest for DNA structure-2— Around 1950, assault on the DNAstructure began in Randall's laboratoryat Cambridge University.— Solving DNA structure is frequentlyattributed to Watson and Crick, but infact it was a gradual and difficultprocess, 90% of which wasaccomplished by crystallographers.— What we remember is the spectacular"discovery", but the betrayal and dramathat surrounded it remains in the shadeand darkens the excitement.The quest for DNA structure-3— X-ray crystallography of simple moleculeswas all the rage at that time, but DNA was notonly big, but like all filamentous molecules, wasimpossible to crystallize!— Instead, a graduate student of Bernall, SvenFurberg, solved the crystal structure of anucleoside cytidine, finding that sugar wasperpendicular to the base.— Therefore, Furberg proposed a model ofDNA in 1952, based on a theoretical model ofpolycytosine monophosphate, with a singlespiral strand, base stacking and sugars parallelto the axis of the spiral (in contrast to Astbury’sproposal, where sugars and bases were in thesame plane).Wool (alpha-keratin) Silk (beta-keratin)The quest for DNA structure-4— A postdoc of Randall, Maurice Wilkins,developed methods to make DNA intofibers, but the fibers, because they had tobe thin (30 µM), were unstable, givingpoor image quality, and Wilkins was onlyable to conclude that pure DNA is indeed aspiral.—To get around the problem of DNA fiberinstability, Wilkins tried X-raying cells,packed with DNA (like sperm cells), butimage quality was still a problem (mostlyfor a different reason now, — becauseDNA in cells is never “pure”), so Wilkinsagain just confirmed that “natural” DNAhas the same spiral structure.The quest for DNA structure-5— Since the image quality precludedWilkins from discerning more detailsabout DNA structure, he eventuallylost interest in the project, and Randallhad to hire another postdoc for thetask.— This time, he was fortunate tostumble upon a new talent in thecrystallography field, RosalindFranklin.— Franklin modified Wilkins’ method,by employing constant humiditychambers to stabilize DNA filamentsby high humidity, and used itsuccessfully to solve the DNAstructure.The quest for DNA structure-6— Specifically, Franklin observed two forms of DNA: A-form at the 75%humidity, and B-form at the ≥90% humidity.— A-form gave pictures with a lot of reflexes, while the more biologically-relevant B-form gave pictures with a few reflexes, appearing much less complex.— Franklin, being a crystallographer, naturally concentrated on solving thestructure of A-form (which is similar to the current structure of DNA-RNAhybrids) and only touched on B-form.A-form B-formThe quest for DNA structure-7— At this point, the influentialtheoretical attempt of Linus Pauling atsolving the DNA structure should bementioned.— Pauling, of course, was the famousprotein crystallographer who proposedthe structure of alpha-spirals and beta-sheets, the two secondary structureelements that form the basis of anyprotein tertiary structure.— The underlying philosophy of bothsecondary polypeptide structures ofPauling was the centrally-locatedbackbone of the polypeptide repeats,with the variable parts of amino acidsfacing outward.The quest for DNA structure-8— That logic seemed to fit perfectlybiopolymers made of compositemonomers, every such monomer havingthe constant part that polymerizes and thevariable part that imparts uniqueproperties on this monomer.— The rationale behind these structures,“tuck in the backbone, stick out thevariable determinants”, makes perfectbiochemical sense for proteins.The quest for DNA structure-9— In fact, it seemed so naturally logical, that Paulinggot epiphany and without any experimental evidenceproposed a remarkable “biochemical” model of DNA.— In this model, three sugar-phosphate backbonesintertwined in a spiral braid, with the bases sticking outin all directions (the “branch-and-leaves” arrangement).— It was a sequel to his famous polypeptide alpha-spiral and a beauty to behold.The quest for DNA structure-10— However, Franklin's painstakingcalculations were revealing a totallydifferent structure.— For one, she calculated that bothA- and
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