2024S1-Lecture-01-updated.pdf

MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM SC/BIOL 3110, 2024 S1 Lecture 01: Introduction to Molecular Biology DNA = Molecule of inheritance Structure and properties of DNA and RNA 1 MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM SC/BIOL 3110, 2024 S1 Email contact: All course-related communication...

MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM SC/BIOL 3110, 2024 S1 Lecture 01: Introduction to Molecular Biology DNA = Molecule of inheritance Structure and properties of DNA and RNA 1 MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM SC/BIOL 3110, 2024 S1 Email contact: All course-related communication, including questions related to course material, or communications regarding accommodations or missed exams etc., should go through the course-specific email account. Please always include your name and student number in your emails. Questions on course material requiring short answers can be asked via email up to 24 hours before a midterm or final. 2 MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM SC/BIOL 3110, 2024 S1 3 Course set up on eClass: 4 Course set up on eClass: 5 Course set up on eClass: 6 MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM SC/BIOL 3110, 2024 S1 Topics that we will cover: 1. DNA basics: history, chemical composition and physical properties 2. DNA topology 3. RNA structures and functional RNAs 4. DNA synthesis and replication 5. Telomeres and telomerase 6. Methods for studying DNA and molecular biology techniques 7. Genome organization and packaging (prokaryotes and eukaryotes) 8. Organization, dynamics and regulation of interphase genomes 9. Long range chromosomal interactions 10. Epigenetics and chromatin regulation 7 MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM SC/BIOL 3110, 2024 S1 Learning outcomes: Upon successful completion of this course, students should be: Knowledgeable in nucleic acids-related properties and concepts Knowledgeable in DNA-based genomes and how genomes are organized Knowledgeable in how genome organization impacts on various biological processes and functions Knowledgeable in experimental techniques, and interpretation of results Appreciative of the experimental nature of scientific discoveries Appreciative of the inter-connectedness of molecular mechanisms that regulate different biological processes within a cell Able to apply knowledge and critical thinking in exams 8 MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM SC/BIOL 3110, 2024 S1 How to do well in this course: Attend classes in person and practice active learning while taking notes à think about what I say in class and put it in your own words when you write things down. Typically, I start each lecture (except the first) doing a re-cap of the previous lecture – the point is to allow students to have time to digest lecture material and hear it a second time or ask questions to clarify. Therefore, ideally should review lecture material before attending next class and ask questions if something is not clear. You can ask questions before, during or after class, by email or by attending Office Hours. Try to figure out connections among different topics – this helps you to make sense of the material and remember important details. Also allows you to see the consistencies among the many cellular pathways. Pay attention to experimental details (use common sense to judge what is important) and understand the reasons and purposes of specific steps. 9 MOLECULAR BIOLOGY I: NUCLEIC ACID METABOLISM SC/BIOL 3110, 2024 S1 Tips on writing exams: Read questions carefully during exam and choose best option that answers the question. With in-person exams, you are able to skip ahead to easier questions and then come back to the harder questions. For multiple choice questions, do not jump and choose answers that are correct statements but do not answer the questions asked. Eliminate obvious incorrect choices and then think about which of the remaining choices answer the questions asked. For written answer questions, read and think about the questions and only provide answers for what was asked for. DO NOT just write down everything you know about a topic. Written answer questions are broken into parts that ask for specific details/answers à meant to guide you to think through the different aspects of the topics 10 Some “fun” facts that we will discuss in this course: 1. Did you know: the first biochemically purified DNA came from pus? 2. What are Twist and Write and how do they relate to cancer-treating drugs? 3. Do humans have the biggest genomes? Does size matter? 4. 3D puzzle: How do you cram 2 meters of DNA into a 10 µM diameter? 5. Does drinking red wine help you live longer? 6. How come calico cats are almost always female and how does that connect to a possible cure for Down syndrome? 7. Does a pregnant mother’s diet pre-determine the weight of the child 30 yrs later? Is “transgenerational inheritance” a real thing? 8. Can we use a chemical that blocks a protein binding to the genome as a male contraceptive drug? 11 What is Molecular Biology? Oxford Dictionary definition: the branch of biology that deals with the structure and function of the macromolecules (e.g. proteins and nucleic acids) essential to life. Wikipedia definition: study of molecular underpinnings of the processes of replication, transcription, translation and cell function. The central dogma of molecular biology where genetic material is transcribed into RNA and then translated into protein 12 What is Molecular Biology? 13 What is Molecular Biology? = Recombinant DNA technology 14 What is Molecular Biology? = Recombinant DNA technology 15 What is the molecule of inheritance? John Lennon Julian Lennon The concept of “likeness” between generations has fascinated philosophers and scientists since the ancient times. Greek scholars such as Pythagoras, Plato, Aristotle have different theories of inheritance. There must be “information” that is passed from one generation to the next. 16 What is the molecule of inheritance? What criteria must be fulfilled by the carrier of inheritance? 17 What is the molecule of inheritance? Gregor Mendel (Austria): Spent 8 years meticulously studying, recording and quantifying crossbreeding of 22 varieties of peas. Published paper titled "Experiments in Plant Hybridisation" in 1866. 18 What is the molecule of inheritance? Mendel’s Laws of inheritance: 1. Law of independent assortment: Specific traits (e.g. stem length or seed colour) operate independent of one another. 2. Law of independent segregation: Inherited characteristics exist in alternative forms (e.g tall vs short) – known as alleles 19 For each characteristic, an individual possesses two paired alleles – one inherited from each parent These pairs segregate in germs cells and recombine during reproduction Each parent transmit one allele to each offspring What is the molecule of inheritance? Mendel’s Laws of inheritance: 3. Law of dominance: For each characteristic, one factor is dominant and appears more often, in a 3:1 ratio The alternative form is recessive The constant 3:1 ratio represents the random combination of alleles during reproduction. Any combination that includes the dominant allele will express the dominant trait Punnett square 20 What is the molecule of inheritance? Gregor Mendel (Austria): In modern terms, Mendel’s factors that carried the traits = genes Also in 1866, Ernst Haeckel (embryologist in Germany) proposed that the nucleus contained the factors responsible for the transmission of hereditary traits 21 What is the molecule of inheritance? Johann Friedrich Miescher (Switzerland/Germany): First to “isolate” what we now know as DNA (1869). Hoped to unravel the fundamental principles of the life of cells by studying leukocytes collected from pus on fresh surgical bandages. In pus, he found the ideal base material for his analyses, and its “histological purity” allowed him to purify the chemical building blocks that constitute cells. http://www.dnaftb.org/ 22 What is the molecule of inheritance? Johann Friedrich Miescher (Switzerland/Germany): Through simple extraction of cells using alkaline solution à nuclei precipitated out of solution à used to isolate chemical substance within nuclei. He named the material found inside the nuclei “nuclein”. By chemical analyses, Miescher determined that nuclein does not contain proteins since he found that it did not contain any sulfur (found in proteins), but contain large amounts of phosphorous. Glass vial containing “nuclein” purified by Miescher 23 What is the molecule of inheritance? Johann Friedrich Miescher (Switzerland/Germany): Later discovered that salmon sperm proved to be the ideal source material for isolating large quantities of pure nuclein. Found that nuclein contained mostly carbon, nitrogen, hydrogen, oxygen, and phosphorous atoms. He was lucky to have picked these cell types for his studies because both leukocytes and spermatozoa are easily purified, and contained mostly nuclei (small cytoplasm) à facilitated enrichment of the nuclear components Modern day picture of collecting salmon sperm 24 What is the molecule of inheritance? Johann Friedrich Miescher (Switzerland/Germany): Nuclein was later renamed “nucleic acid” (by Richard Altman in 1889) when it was discovered to have properties of acids. In Miescher’s time, the vast majority of scientists were convinced that the more complex proteins must be the carriers of genetic information. Why? 25 What is the molecule of inheritance? Albrecht Kossel (Germany): Worked on the constitution of the cell nucleus – in 1880s, showed that nuclein consisted of a protein component (which he termed “histon”, i.e. modern day histones), as well as a non-protein component (nucleic acids). Discovered that nucleic acids contain four nitrogen-containing groups: cytosine, thymine, adenine, and guanine. Proteins were made up of 20 amino acids whereas nucleic acids only has 4 different bases à at the time, thought to be “not complex” enough to be molecule of inheritance 26 What is the molecule of inheritance? Walter Flemming (Germany): In the 19th Century, scientists started to use microscopy to study cells, cell functions and structures. However, resolution of the microscopes was poor and the cells’ inner structures are mostly grey-on-grey shapes. In 1879, Flemming found a basophilic dye that specifically stained the material inside the cell nucleus, which he termed “chromatin” (Greek word for colour). Using this dye to stain salamander embryos going through cell division, Flemming saw the stained material (chromatin) collect into thread-like structures (chromosomes, from Greek word meaning coloured bodies). 27 What is the molecule of inheritance? Walter Flemming (Germany): He also coined the term “mitosis” (Greek word for threads) to describe the behaviour of chromosomes during cell division. During mitosis, he saw that each individual chromosome break into two, thus doubling the number of chromosomes. He also saw that identical sets of chromosomes being pulled apart and separately go to the two dividing cells. Therefore, each daughter cell contained a set of chromosomes identical to the parental cell 28 What is the molecule of inheritance? William Bateson (USA) and Wilhelm Johannsen (Denmark): In 1905, Bateson first coined the term “genetics” (Greek word meaning to give birth) to describe the study of inheritance. In 1909, Johannsen introduced the basic terminology for genetics: Genes = units of hereditary information Genotype = genetic constitution of an organism Phenotype = an organism’s totality of inherited characteristics Relationship between genotype and phenotype 29 What is the molecule of inheritance? Thomas Hunt Morgan (USA): Embryologist turned heredity researcher– starting from 1907, pioneered the breeding of the common fruit fly (Drosophila melanogaster) for genetic studies. Found that while almost all Drosophila has red eyes, very rarely there are a few that have white eyes (in one breeding experiment, out of 1,237 first generation offsprings, 3 had white eyes). Noticed that all white-eye flies were male, and proposed that association of eye color and sex in fruit flies had a physical and mechanistic basis in the chromosomes. Red- and White-eyed Drosophila 30 What is the molecule of inheritance? Thomas Hunt Morgan (USA): Discovered sex-linked genes, and developed the concept of genetic linkage (i.e. genes that are close together on the chromosomes are often inherited together during meiosis à contradicts Mendel’s first law). He also used physical, chemical, or radiological means to mutate Drosophila and used breeding experiments to follow heritable mutations. The physical basis of genetic linkage is how close the genes are to one another on a chromosome (or on different chromosomes) 31 What is the molecule of inheritance? Hermann Muller (USA): Worked with Hunt to use X rays to induce mutations in Drosophila genome. Found that genes can be mutated by chemical or radiological insults, and more importantly, the mutated genes can be passed on from one generation to the next! I.e. genes can be inherited What do genes do? 32 What is the molecule of inheritance? George Beadle and Edward Tatum (USA): Used bread mould, Neurospora crassa, to determine if and how genes control known biochemical reactions. Neurospora was chosen because a) it was easy to grow, b) it was easy to produce and observe mutation effects (since haploid state only has one set of unpaired chromosomes), and c) the biochemistry of amino acid synthesis was already known. Goal was to understand connection between genes and phenotypes More specifically, used genetic screens to identify genes responsible for making specific amino acids Lab cultures of Neurospora in flasks 33 Types of genetic screens: Common approaches to identify gene functions: Random mutagenesis Targeted mutagenesis 34 What is the molecule of inheritance? George Beadle and Edward Tatum (USA): For their classic experiment (published in 1941), they first irradiated a large number of Neurospora haploid spores and tested their growth on various growth medium... ➔ Perform FORWARD genetic screen experiment: ➔ Mutate spores and screen for loss of ability to synthesize a specific amino acid (i.e. lost the ability to grown in minimal medium) Growth: WT: mut: P P P O Medium not supplemented with any amino acids Minimal medium plus the amino acid indicated 35 What is the molecule of inheritance? Neurospora (bread mould) life cycle: 36 What is the molecule of inheritance? George Beadle and Edward Tatum (USA): By this method they isolated four strains of Neurospora that only grew on minimal medium if supplemented with arginine. Genetic analyses showed that each strain differ from the normal wild type by one gene Suggested that loss of multiple genes resulted in same phenotype à loss of the production of a single amino acids (e.g. arginine). One gene one enzyme hypothesis: DNA Protein The one gene, one enzyme (or later one gene, one polypeptide) concept is considered by some as the pre-cursor of the Central Dogma. 37 What is the molecule of inheritance? To test hypothesis: 38 What is the molecule of inheritance? The carrier of heredity must fulfill these following criteria at the molecular level: Molecule of inheritance = Gene But what are genes made up of? 39 DNA = molecule of inheritance Frederick Griffith (UK): Bacteriologist interested in epidemiology and pathology of bacteria causing pneumonia. 40 DNA = molecule of inheritance Frederick Griffith (UK): Discovered transformation in Streptococcus pneumoniae (1928) à experiment suggested that heritable traits can be exchanged between bacterial strains. Live and virulent bacteria can be recovered from dead mouse’s blood Transforming principle: non-virulent strain transformed into virulent strain 41 DNA = molecule of inheritance Avery, MacLeod and McCarty (USA): What is the material responsible for Griffith’s transforming principle? Which one produced virulent bacteria? 42 DNA = molecule of inheritance Avery, MacLeod and McCarty (USA): In 1944, proved that material responsible for transformation = DNA! i.e. DNA = genetic material in bacteria 43 DNA = molecule of inheritance Alfred Hershey and Martha Chase (USA): What is the genetic material in bacteriophage? DNA or protein? 44 DNA = molecule of inheritance Alfred Hershey and Martha Chase (USA): Famous blender experiment done in 1952: 45 DNA = molecule of inheritance Alfred Hershey and Martha Chase (USA): Famous blender experiment done in 1952: 46 DNA = molecule of inheritance Alfred Hershey and Martha Chase (USA): Famous blender experiment done in 1952: Phage coats contain 80% of 35S label Infected bacteria contain 70% of 32P label 47 DNA = molecule of inheritance Alfred Hershey and Martha Chase (USA): This experiment directly showed that only the DNA of the parent phages enters the bacteria and becomes part of the progeny phages à i.e. genetic material of bacteriophages = DNA More importantly, the progeny phages contain 30% of 32P label and < 1% of 35S label 48 DNA = molecule of inheritance What is DNA? DNA = deoxyribonucleic acid DNA is a polymer of nucleotides A nucleotide consists of pentose sugar nitrogenous base phosphate group 49 DNA = molecule of inheritance Sugar: Pentose = sugar molecule with 5 carbon atoms Ribose = single-ring pentose sugar. The number of carbon atoms runs clockwise Furanose = 5-member ring consisting of 4 carbon atoms and 1 oxygen atom Deoxyribose vs ribose sugars: DNA vs RNA missing -OH (alcohol) group 50 DNA = molecule of inheritance Nitrogenous base: Can either have a pyrimidine or purine ring Pyrimidine = aromatic compound with 2 N at C1 and C3 of a 6-membered ring Purine = aromatic compound consisting of a pyrimidine ring and an imadazole ring (C3H4N2) 51 DNA = molecule of inheritance Nitrogenous base: Pyrimidine = cytosine (C*), thymine (T), or uracil (U) Purine = adenine (A) or guanine (G) *Note that cytosine can be methylated at the C5 position = m5C 52 DNA = molecule of inheritance Nitrogenous base: Pyrimidine = cytosine (C*), thymine (T), or uracil (U) Purine = adenine (A) or guanine (G) Spontaneous deamination turns 5mC to T à i.e. spontaneous mutagenesis! 53 Side note: meC G mismatch T G not repaired repaired C G T G DNA replication T A permanent mutation C G 54 DNA = molecule of inheritance Sugar + nitrogenous base = nucleoside Covalent bond between the D-ribose and nitrogenous base = b-glycosidic bond Nucleosides can be in syn or anti conformations Syn: base is over furanose ring Anti: base is not over furanose ring The syn conformation is generally sterically unfavourable; however, this conformation is found in the Z-form of DNA 55 DNA = molecule of inheritance Sugar + nitrogenous base + phosphate = nucleotide Phosphate group is joined to the C-5’ position of the nucleoside by a phosphoester bond. Nucleotides can have one, two, or three phosphate groups and are called NMPs, NDPs, and NTPs respectively. 56 DNA = molecule of inheritance DNA = long polymer chains of nucleotides Nucleotides are linked by a phosphodiester bond between the phosphate group at the C-5' position and the OH group on the C-3' position. The phosphodiester linkage create the repeating, sugar phosphate backbone of the polynucleotide chain. 57 DNA = molecule of inheritance DNA = long polymer chains of nucleotides A Because the phosphate links the C-5’ and C-3’ of two nucleotides, this confers directionality to the DNA polymer. B DNA and RNA sequences are conventionally written 5’ to 3’ (direction of synthesis) 58 DNA = molecule of inheritance Erwin Chargaff (Austria/USA): Prior to Chargaff, the mainstream theory suggested that DNA was made up of equal amounts of the 4 bases = Tetranucleotide hypothesis. Chargaff used the newly developed paper chromatography and UV spectrophotometer techniques to revisit this issue. Collected DNA from various organisms, hydrolyzed the phosphodiester bonds using acid to yield individual nucleotodies, and analyzed the base compositions. 59 DNA = molecule of inheritance Erwin Chargaff (Austria/USA): Experimental results: Noticed [A] = [T], and [G] = [C] 60 DNA = molecule of inheritance Erwin Chargaff (Austria/USA): Experimental results: * Noticed [A] = [T], and [G] = [C] * With some exceptions = Szybalski's rule 61 DNA = molecule of inheritance Erwin Chargaff (Austria/USA): Chargaff’s two rules: 1. The nucleotide composition of DNA varied from one species to another. i.e. there is a lot more variability in DNA than previously thought. 2. For almost all DNA, the amount of adenine (A) is equal to the amount of thymine (T), and the amount of guanine (G) is equal to the amount of cytosine (C). In other words, the total amount of purines (A + G) and the total amount of pyrimidines (C + T) are usually equal. à This rule has direct implications for determining the structure of DNA 62 What is the structure of DNA? Other events that were important to the elucidation of the structure of DNA In 1937, William Astbury performed the first X-ray diffraction experiment on DNA and concluded that DNA has a regular structure. In 1938, he also determined that DNA has a 0.34 nm spacing. ~ 1951, Maurice Wilkins and Rosalind Franklin used X-ray diffraction to study the Band A-forms of DNA Basic scheme of X-ray diffraction to solve the structure of DNA 63 What is the structure of DNA? Other events that were important to the elucidation of the structure of DNA By the end of 1951, Franklin and Wilkins were convinced that the B-form of DNA has a helical structure. By Jan of 1953, Franklin had resolved some of her previous conflicting data, and started to write 3 manuscripts, 2 of which suggested a double helical backbone for DNA. Unbeknownst to Franklin, Wilkins had shown Franklin’s famous photograph 51 to Jim Watson, which gave Watson and Crick the insight needed to solve the structure of DNA. Photo 51, taken in 1952 This photo allowed Franklin to determine that each turn of the helix in the B form is 34 Å long and contains 10 base pairs separated by 3.4 Å each 64 What is the structure of DNA? Linus Pauling (USA): In addition to Wilkins, Franklin, Watson and Crick, Linus Pauling was also in hot pursuit of the structure of DNA. Pauling was a chemist who, in the 1930s, made great contributions to the understanding of nature of chemical bonds between atoms of molecules (won Nobel Prize in Chemistry in 1954 for this work). He had an interest in the structure of how polypeptides fold and, in 1948, deduced the structure of the alpha helices using paper to construct models à directly influenced Watson and Crick’s approach of using models to solve structures. Reconstruction of Pauling’s paper, used to construct the model of an alpha helix 65 What is the structure of DNA? Linus Pauling (USA): Because Pauling used the poor resolution X-ray diffraction data from Astbury, and because of his incorrect assumption that the phosphate backbone of DNA could form the core of the DNA molecule, he came up with a triple helix model, which would have been unstable and thus incorrect. Linus Pauling’s actual notes from Nov 1952 66 What is the structure of DNA? James Watson and Francis Crick (USA/UK): In 1951, Watson joined the Cavendish laboratory at Cambridge and worked with Crick to solve the structure of DNA. Influenced by Pauling’s success in using modeling, Watson and Crick took the same approach to construct a model that could explain all the features of DNA known then. 67 What is the structure of DNA? Watson and Crick and the double helix: For example, at that time, the chemical composition of DNA and the chemical structures of the nucleotides were known. Chargaff already showed that there were equal numbers of A:T and C:G in almost all DNA. In 1952, Alexander Todd showed that nucleotides of DNA chains are held together by 3' à 5' phosphodiester bonds (i.e. DNA has directionality). Finally, the X-ray diffraction patterns obtained by Astbury and Franklin showed that DNA had a constant width and likely helical in structure. Question is: how does everything fit together? 68 What is the structure of DNA? Watson and Crick and the double helix: Model building allowed the relative positioning of the various atoms to be checked, to ensure that pairs of groups that formed bonds were not too far apart First, Watson and Crick thought that the bases paired in a “like with like” fashion (e.g. A-A) However, that would not produce a uniform width… For example: 69 What is the structure of DNA? Watson and Crick and the double helix: They therefore concluded that a purine always has to be paired with a pyrimidine to obtain uniform width. Moreover, to explain Chargaff’s rules, they concluded that A can only pair with T, and C can only pair with G: 70 What is the structure of DNA? Watson and Crick and the double helix: Finally, through model construction, the found that an anti-parallel orientation of the nucleotide chain backbones worked best to orient the base pairs in the centre of a double helix 71 What is the structure of DNA? Watson and Crick and the double helix: Note: this structure differs from Pauling’s idea that the phosphate backbone of DNA lined up in the centre of the helix (which was W&C’s initial assumption as well). 72 Properties of DNA structure: Key features of the double helix: The two strands are complementary but not identical. For example: 5’ GAATCCTATTGCATC 3’ 3’ CTTAGGATAACGTAG 5’ 5’ GAATCCTATTGCATC 3’ 3’ CTTAGGATAACGTAG 5’ 73 Properties of DNA structure: Concept of complementarity: The concept of complementarity has huge implications on how genetic material can be copied during duplication. It means that one strand can serve as template for the synthesis of the other strand. As Watson and Crick wrote in their 1953 paper: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” 74 Properties of DNA structure: Concept of complementarity: Base pairing and complementarity allowed genetic information to be copied. 75 Properties of DNA structure: Concept of complementarity: Base pairing and complementarity allow genetic information to be copied during DNA replication. 76