MCB 502A-2014. Lecture #2DNA Structure — The quest for the DNA structure— 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 DNA structure-1What was known about DNA structure before 1950:1. In the 40s: DNA could not be a tetranucleotide, is likely long linear chain... 2. Bill Astbury: first DNA crystallography (1938) => DNA is densely packed. Astbury proposed the first model of DNA structureDistance between nucleotides is 3.4Å.3. The mysterious Chargaff rule (A=T, G=C). William Astbury0.34 nm"pile of pennies"sugar baseBy the way…Bill Astburry was the first to use the term "Molecular Biology” in his 1950 Harvey Lecture.— Scientists objected, of course, … — In Europe of that time, laboratories had their own names…—University called it "the Department of Biomolecular Structure". Committee argued that "he may know something about molecules, but he knows precious little about Biology…" — Astbury <…> proposed the term "Molecular Genetics" in 1961! Astbury at his Department of Textile Physics at the University of Leeds.The quest for DNA structure-2— Randall's laboratory Cambridge ~1950 began the quest for the DNA structure. — Solving DNA structure: Watson and Crick (?)A gradual and difficult process, 90% of which was pre-Watson and Crick. — The spectacular "discovery", darkened bydrama and betrayal.The quest for DNA structure-3— …all the rage at that time, but DNA was <…> impossible to crystallize! — Sven Furberg solved a crystal structure of a nucleoside cytidine …. — Furberg proposed the second model of DNA (1952), based on a theoretical model of polycytosine monophosphatea single spiral, base stacking and sugars parallel to the axis of the spiral…Wool (alpha-keratin) Silk (beta-keratin)Sven FurbergThe quest for DNA structure-4—Maurice Wilkins developed methods to make DNA into fibers, <…> concluded that pure DNA is indeed a spiral. — ….also X-rayed cells, packed with DNA (like sperm cells), <…> and confirmed that “natural” DNA has the same spiral structure.The quest for DNA structure-5— Wilkins lost interest…, and Randall had to act. —a new talent in the crystallography field, Rosalind Franklin. — Franklin modified Wilkins’ method, <…> and solved the overall DNA structure.The quest for DNA structure-6—Franklin observed A-form at the 75% humidity, B-form at the ≥90% humidity. — A-form had a lot of reflexes, while the B-form gave pictures with a few reflexes. — Franklin decided to concentrate on the A-form <…> and only touched on B-form. A-form B-formThe quest for DNA structure-7—Linus Pauling proposed a third model pf DNA structure. — Pauling proposed alpha-spirals and beta-sheets…. — The underlying philosophy of both secondary polypeptide structures was… The Nobel Prize in Chemistry 1954The quest for DNA structure-8— That highly-intuitive logic seemed to fit perfectly biopolymers made of composite monomers, … — A perfect rationale for proteins…The quest for DNA structure-9— Pauling:“biochemical” model of DNA. — In this model, <…> (the “branch-and-leaves” arrangement). — a sequel <…> and a beauty…The quest for DNA structure-10— Franklin's painstaking calculations… — 1) likely double-helices, … — 2) sugar-phosphate backbone must run on the outside in a wide spiral, …— 3) deoxyriboses parallel… Linus Pauling:“The way to get good ideas is to get lots of ideas, and throw the bad ones away.” Pauling’s model Franklin’s modelThe quest for DNA structure-11— The two groves…, the overall diameter… — <…> the two DNA strands run antiparallel. — <…> most likely for B-DNA as well...The quest for DNA structure-12— Franklin was <…> not in a hurry … — Both to her credit and to her detriment, … — <…> Wilkins was sharing Franklin’s results <…> — a bizarre turn....The quest for DNA structure-13— The main sympathizers <…> regarded as marginals. — Francis Crick… — Jim Watson … … and, no wonder… Jim Watson and Francis CrickThe quest for DNA structure-14— What Watson had plenty… — The world was waiting … — Watson was ready <…> but lacked the tools... — Somebody had to give Watson a crush course in crystallography …The quest for DNA structure-15— The disgruntled Wilkins… — … ego tickled by the obvious inferiority... — In economic terms, … — … Watson and Crick proved…The quest for DNA structure-16— <…> Franklin’s developing model + critical detail: complementarity of the bases in the two strands! — <…> W+C correctly guessed <…> the right-handed helix. — The paper of Watson and Crick:the “genetic” model of DNAexplained how the gene replicatesMolecular Biology was born!The quest for DNA structure-17— Franklin at that time .... — The bases were always “disordered”… — <…> Franklin wanted to grasp the overall shape and structure of this beautiful double spiral <…>.The quest for DNA structure-18— One can speculate <…> to account for the constant diameter of the DNA double helix <…> — <…> unlikely to arrive at the correct base pairing pattern right away. — At that time <…> the enol tautomeric forms over the keto forms <…>.The quest for DNA structure-19— <…> enol-guanine <…> pairs perfectly with keto-thymine…. — <…> enol-thymine <…> is a better match to keto-guanine. — The fact that DNA bases prefer keto-forms …The quest for DNA structure-20— Watson and Crick <…> shared office with Jerry Donohue, <…>— Once W+C were forced to have DNA bases in the keto forms, …— This was a breakthrough! — <…> combinations:— explained the mysterious Chargaff’s rule (A = T, G = C in duplex DNA); — provided the means to replicate the genetic material! Jerry DonohueThe quest for DNA structure-21— <…> the A-T, G-C pairing could have never been solved by crystallography of natural DNAs <…> —<…> the only way to arrive at it at that time was molecular modeling <…>— In other words, the structure of the
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