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The Discovery, Structure, and Function of DNA The Link from Function to Form 1869: Friedrich Meischer isolates DNA (“nuclein”) from fish sperm and human pus. 1900: Hugo de Vries, Carl Correns, and Erich von Tscermak rediscover Mendel’s ratios. The big debate: Can Mendelian mechanics measure evolutionary change? Problems with replicating Mendel’s simple rules using different organisms and traits lead to a renewed interest in locating the “gene” and describing its physio-chemical dynamics. 1919: Thomas Morgan demonstrates conclusively that genes are physical units lying along chromosomes, which in turn lie inside every cell of every organism. 1943: Oswald Avery isolates deoxyribonucleic acid (DNA) as the chemical manifestation of heredity. Discovery of DNA: The Players James Watson: Fresh Indiana U. PhD, starts postdoc at Cambridge’s Cavendish Labs under the direction of Sir Lawrence Bragg Francis Crick: Currently working at Cavendish on X-ray diffraction of proteins Maurice Wilkins, Rosalind Franklin: Working at X-ray diffraction of DNA at London’s King’s College Linus Pauling: Working at Caltech on the structure of proteins Discovery of DNA: The Game The Sides: Those who believed that proteins held the key to genetic traits and that these traits were transferred biologically, and those who believed that DNA held these traits, and the transfer was chemical. 1951: Pauling predicts alpha-helix structure of collagen proteins, using model-building techniques. Wilkins shows X-ray diffraction pictures of DNA. DNA is known to be • crystalline (regular repeating structure) • acidic (electron accepting) • contains one or more a sugar-phosphate backbones • backbone attached to bases composed of adenine (A), cytozine(C), guanine(G), and thymine(T), with A-T and G-C pairs appearing in approximately equal quantities. But how is it constructed? Discovery of DNA: The Play 1952: Watson and Crick set out specifically to win the Nobel prize by finding the structure of DNA. Main questions: How many backbone strands are there, and are they centrally located or on the outside of the molecule? Solution technique: Make molecular models out of metal, try to fit the components of the molecule together to satisfy X-ray diffraction data. First try: Three central strands in a helical shape. Wilkins&Franklin observe that resulting water content is too far off. Back to the drawing board. Discovery of DNA: The Chase 1953: Pauling postulates similar 3-chain central strand model for DNA. Watson&Crick observe chemical discrepencies. Visit to Wilkins&Franklin confirms outside helix model. Watson&Crick begin on outside chain helix, choose 2-strand model. Problem with outside model: How do you fit the ACGT molecules inside the helix, and still satisfy the correct molecular distances in the backbone? Second try: 2-strand outside model, with AA, C-C, G-G, T-T matching along the inside. Problems with fit. Final form: A-T, G-C pairing satisfies chemical laws, X-ray data, and are symmetric. February 28, 1953: Crick announces to a Cambridge pub that they had “found the secret of life”. Structure and Function of DNA DNA (deoxyribonucleic acid): Found in every cell in an organism in exact copies (except for the gametes) The Structure of DNA: Two helically intertwined backbones made up of alternating phosphate and deoxyribose (sugar) molecules supporting internal base pairs adenine (A) paired with thymine (T) cytosine (C) paired with guanine (G) The pairs can occur in any combination and order as necessary to define the organism. The Structure of DNA Replication of DNA During Cell Division: Mitosis (Interphase) 1. Enzymes unzip the DNA molecule between its base pairs, leaving two complementary single strands. 2. DNA polymerase matches the bases in each strand, attaching new complementary strands, creating two new copies of DNA. 3. The two strands separate and go to each copy of the replicated cell. Replication of DNA during mitosis Replication of DNA to Produce Gametes: Meiosis Steps of meiosis: 1. Before meiosis, the nucleus contains two copies of each chromosome, a maternal and a paternal copy. This is called a homologous chromatid pair. 2. In the nucleus, the chromosome pairs separate, and each side replicates itself exactly. The identical copies join to form two pairs, called sister chromatids, of each chromosome. 3. The two pairs line up, and may swap pieces of chromosome between either of the maternal and paternal members. This exchange is called crossing over. This is the first way of realizing of Mendel’s law of independent assortment. 4. Each pair of a chromosome is then drawn to opposite sides of the nucleus, and the nucleus splits into two nuclei, each with complete paired copies of the genome. Which pair goes to which nucleus is the second way of realizing Mendel’s law of independent assortment. 5. The paired chromosomes in each nucleus now separate and are drawn to opposite sides of the new nucleus. The nucleus splits again, forming four gametes, each containing one copy of each chromosome. This is the way of realizing Mendel’s law of independent segregation. Steps of Meiosis More on Crossing Over Two different halves of the replicated chromosome (called cromatids) swap genetic material by the following steps : 1. They are cut at the same place on the DNA molecule. 2. A section of DNA from each member of the sister pair crosses over to match with the same section of DNA from the homologous chromatid, forming a connected pair of homologous chromatids called a Holliday junction. This will involve some “repairing” of mismatched base-pairs in order to make complementary copies across the new DNA molecule. 3. One of the two appropriate backbone strand pairs are then cut at the opposite ends of the crossing region, and reconnected to the matching backbone of the DNA strand that it will now become part of. This creates two new complete chromatids. It can be done a number of times, and then the chromatids are matched with a mixed or nonmixed partner in the meiosis process. Reconnect Repair these mismatched sections cross over in this section cut at these two points original chromatid pair . I OR II resulting chromatid pair (choose one) The Crossing Over Process I:these OR II: these A T T A G C T A A T A T A T T A G C T A A T A T A T T A G C T A A T A T G C C G A T C G A T A T G C C A A A C G G T T T GC T A AT CG AT AT G C A T T A C G G C A T G C A T T A C G G C A T G C A T T A C G G C A T A T T A G C A T T A T A I GC T A AT A T T A T A G C A T T A G C T A T A A T T A T A A T T A G C G C C G T A II A T T A G C GC T A AT G C A T T A I GC TA TA G C C G T A A Small Example OR GC TA TA G C C G T A II GC T A AT G C A T T A Proteins Proteins are the basic functional units in any organism. They are made up of a single carbonnitrogen backbone with docking points each supporting one of 20 amino acids. Amino acids can occur in any combination and order as necessary to define the protein. From DNA to Protein • The protein amino acid sequence is coded in a specific piece of the DNA called a gene. • The gene is made up of triples of base pairs, called codons, each of which corresponds to a specific amino acid. • Each gene has associated with itself a promoter sequence of DNA upstream of the gene sequence, and a termination sequence downstream, which identifies its location in the genome. • The gene sequence may also have intron portions which do not code any part of the protein, and must be removed before protein production starts. The 20 Amino Acids amino acid alanine arginine aspartic acid asparagine cysteine glutamic acid glutamine glycine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tryptophan tyrosine valine 3-L abbrev. Ala Arg Asp Asn Cys Glu Gln Gly His I le Leu Lys Met Phe Pro Ser Thr Trp Tyr Val 1-L abbrev. A R D N C E Q G H I L K M F P S T W Y V Map of Codons to Amino Acids Each mRNA triple codes for either an amino acid or a stop codon (TC). first base U C A G second base U C A G Phe Ser Tyr Cys Phe Ser Tyr Cys Leu Ser TC TC Leu Ser TC Trp third base U C A G Leu Leu Leu Leu Pro Pro Pro Pro His His Gln Gln Arg Arg Arg Arg U C A G Ile Ile Ile Met Thr Thr Thr Thr Asn Asn Lys Lys Ser Ser Arg Arg U C A G Val Val Val Val Ala Ala Ala Ala Asp Asp Glu Glu Gly Gly Gly Gly U C A G The Central Dogma DNA → RNA → protein Perhaps the most important process in cellular biology is the process that changes a gene into a protein. This process is called the expression of that gene, and the process by which this is done is call the central dogma. RNA: Intermediary in the copying of DNA to either itself (replication) or to proteins The structure of RNA: A single sugar-phosphate backbone with single bases attached. The bases are the same, except that thymine is replaced by uracil (U), which likewise matches with a T in replication. The Production of a Protein Initiation: The production of a protein begins when a protein called a transcription factor binds onto a promotor site of the DNA. Transcription: RNA polymerase then goes to work at the promoter site, and moves along the DNA strand, producing a complementary strand of messenger RNA (mRNA), except that U matches with A. When the process reaches a certain termination sequence, the process halts and the mRNA is passes out of the nucleus to be translated into a protein. (The original strand may have to be additionally processed to remove blocks of introns that do not contribute to the protein.) Translation: A rhibosome (rRNA) matches each codon in the mRNA to a transfer RNA (tRNA) structure, which contains • at one end an anticodon complementary to the mRNA triple, and • at the other end an amino acid which corresponds to the codon indicated by the original DNA triple. The rhibosome brings in the tRNA in succession as it moves down the mRNA strand, hooking the new amino acid to the ever-growing protein. Termination: When the terminator triple is reached (which may not necessarily be the end of the mRNA strand!), the rhibosome lets go of the protein, and it is free to do its assigned job. The Central Dogma Example Suppose a protein is to be made from a strand of DNA with the following base pairs: −→ GGAATDGATCGGCGTCATTTTTGTGCAAGTCTA TAGACTTGCACAAAAATGACGCCGATCGATTCC ←− The arrows indicate the opposite direction in which the bases will be searched on each strand. The protein is identified by promoter site TTGCACA and terminator site ATTCC. This is found on the bottom strand, and the mRNA is produced from that point on. The resulting mRNA strand is UUU|UAC|UGC|GGC|UAG|(C) with the | marking the (complementary) codons of the mRNA strand, and the stop (complementary) codon UAG indicating the end of the protein. (Note that there may be more letters after the stop codon; these are ignored by the protein production process.) The triples in this sequence now match with tRNA structures with paired anticodon-amino acid pairs AAA---Phe, AUG---Tyr, ACG---Cys, CCG---Gly and so the new protein has amino acid residues phenylalanine, tyrosine, cysteine, and glycine, in that order. Note that if the RNA polymerase had slipped one base-pair in the start of the transcription, we would have had mRNA UUU|ACU|GCG|GCU|AGC|UAA which would have produced the protein with amino acids phenylalanine, threonine, alanine, alanine, and serine.