Information Flow in Cells (I) Lecture 8 2024 PDF

Information Flow in Cells (I) Prepared by: AP Dr. Alvin Hee, and AP Dr. Faridah Qamaruz Zaman BGY 3002 Cell & Molecular Biology  Learning Outcomes (1) Upon completing this lecture, you are expected to be able to: 1. Explain the need to have a sec...

Information Flow in Cells (I) Prepared by: AP Dr. Alvin Hee, and AP Dr. Faridah Qamaruz Zaman BGY 3002 Cell & Molecular Biology  Learning Outcomes (1) Upon completing this lecture, you are expected to be able to: 1. Explain the need to have a secure and reliable storage and transfer of cellular information. 2. Describe the basic structural features of the nucleus leading to the DNA double helix. 3. Define the basic terminologies used in this lecture e.g. gene and genome, etc. 4. Explain the chemical nature of the polynucleotides.  Learning Outcomes (2) 5. Explain briefly certain key experiments leading to the discovery of the DNA as the genetic material (e.g. Griffith, Hershey-Chase). 6. Relate the importance of the Watson-Crick DNA model from a functional perspective. 7. Identify the different DNA structures and key characteristics of those structures. 8. Identify the class of enzyme involved in the supercoiling process and state briefly the reasons why supercoiling is important.  Introduction The need for secure and reliable storage and transfer of biological information. Why? - In order for cells to grow and divide; and for species to remain viable. - If a cell is unable to store and pass on important directions/instructions (information) to its progeny, then it makes no difference if a macromolecule has the correct structure or the cell is able to obtain energy. Flow of Information in Cells  Nucleus Chromosomes Chromatins Nucleosomes DNA (gene)  Basic Terminologies Nucleus: The organelle that contains the eukaryotic cell’s genetic material. Chromosomes: Threadlike strands that are composed of the nuclear DNA of eukaryotic cells and are the carriers of genetic information. Chromatin: A complex nucleoprotein material that composes the chromosomes of eukaryotes Gene: A unit of inheritance that governs the character of a particular trait; functional unit of heredity. A segment of DNA containing the information for a single polypeptide. Genome: The complement/ set of genetic information unique to each species. Full DNA sequence of an organism or the full DNA or RNA sequence of a virus. The Genomes of Many Organisms Have Been Sequenced  Personalised genomes The first nearly complete human genomes sequenced were: Craig Venter James Watson A Han Chinese A Yoruban from Nigeria A female leukemia patient Seong-Jin Kim Steve Jobs (paid USD 100,000) As of June 2012, there are 69 nearly complete human genomes publicly available Useful for predictive and preventive medicine  Nucleus of An Eukaryotic Cell The contents of the nucleus are enclosed by a double-membrane nuclear envelope. Chromosomes: extended fibres of chromatin. Nuclear matrix: protein- containing fibrillar network. Nucleoli: sites of rRNA synthesis. Nucleoplasm: fluid in which solutes of the nucleus is dissolved. Nuclear envelope: boundary between the nucleus and the cytoplasm of cell. - two membranes separated by a perinuclear space - two membranes fused at sites forming a circular nuclear pore (Nuclear Pore Complex, NPC: proteins & RNA in and out of nucleus) - inner surface of envelope lined with nuclear lamina (composition - lamins)  Chromosomes Visible only during mitosis. Consists of chromatin fibres, DNA and associated proteins. Each chromosome contain a single, continuous DNA that is packed to occupy a small space. Histones and non-histone chromosomal proteins.  Nucleosomes Lowest level of chromosome organization. Histones: highly conserved between different species. Consists of DNA and histones as repeating units (core particle & linker). DNA wrapped around the core complex. Histone core complex: Two molecules each of histones H2A, H2B, H3 and H4 (octamer formation). Histone H1 located external to nucleosome core particle.  Histone H1 binds to the linker DNA & facilitate packing of nucleosomes into 30 nm fibres. +ve charged residues on histone interact with –ve charged phosphate groups in DNA backbone.  The Nature of Genetic Material Historical Background Miescher isolated nuclei from pus (white blood cells) in 1869 – Found a novel phosphorus-bearing substance = nuclein. – Nuclein is mostly chromatin, a complex of DNA and chromosomal proteins. End of 19th century – DNA and RNA separated from proteins. Levene, Jacobs, et al. characterized basic composition of DNA and RNA.  Transformation in Bacteria Key experiments done by Griffith in 1928. Observed change in Streptococcus pneumoniae — from virulent (S) smooth colonies where bacteria had capsules, to avirulent (R) rough colonies without capsules. Heat-killed virulent colonies could transform avirulent colonies to virulent ones.  Outline of Griffith’s Transformation Experiments DNA: The Transforming Material In 1944 a group used a transformation test similar to Griffith’s procedure taking care to define the chemical nature of the transforming substance – Techniques used excluded both protein and RNA as the chemical agent of transformation. – Other treatments verified that DNA is the chemical agent of transformation of S. pneumoniae from avirulent to virulent.  DNA Confirmation In 1940s geneticists doubted use of DNA as it appeared to be monotonous repeats of 4 bases. By 1953 Watson & Crick published the double-helical model of DNA structure and Chargaff had shown that the 4 bases were not present in equal proportions. Hershey and Chase demonstrated that bacteriophage infection comes from DNA. Procedure for the Hershey-Chase Transformation Experiments  The Chemical Nature of Polynucleotides Biochemists determined the components of nucleotides during the 1940s The component parts of DNA – Nitrogenous bases: Adenine (A) Cytosine (C) Guanine (G) Thymine (T) – Phosphoric acid – Deoxyribose sugar  Nucleotides and Nucleosides RNA component parts – Nitrogenous bases Like DNA except Uracil (U) replaces Thymine – Phosphoric acid – Ribose sugar Bases use ordinary numbers Carbons in sugars are noted as primed numbers Nucleotides contain phosphoric acid Nucleosides lack the phosphoric acid  Purines and Pyrimidines Adenine and guanine are related structurally to the parent molecule purine Cytosine, thymine and uracil resemble pyrimidine  DNA Linkage Nucleotides are nucleosides with a phosphate group attached through a phosphodiester bond Nucleotides may contain one, two, or even three phosphate groups linked in a chain  A Trinucleotide The example trinucleotide has polarity – Top of molecule has a free 5’-phosphate group = 5’ end – Bottom has a free 3’- hydroxyl group = 3’ end  Summary DNA and RNA are chain-like molecules composed of subunits called nucleotides Nucleotides contain a base linked to the 1’-position of a sugar and a phosphate group Phosphate joins the sugars in a DNA or RNA chain through their 5’- and 3’- hydroxyl groups by phosphodiester bonds  X-ray diffraction pattern of DNA fibre suggesting the helical nature of DNA (Rosalind Franklin, 1953) X-ray diffraction of B form of purified DNA fibres. Strong arcs on periphery- closely spaced aspects of the molecule. Inner cross pattern of spots reveal the helical nature of the molecule.  DNA Structure The Double Helix Rosalind Franklin’s X-ray data suggested that DNA had a helical shape. The data also indicated a regular, repeating structure. Watson and Crick proposed a double helix with sugar-phosphate backbones on the outside and bases aligned to the interior. Model of DNA built by James Watson & Francis Crick, Cambridge University 1953. (Nobel Prize, 1962) with Maurice Wilkins 2-34  Co-discoverers of DNA structure ancestry.com 2-35  Johann Friedrich Miescher’s Discovery of Nuclein: It all started humbly enough in 1869, when Swiss chemist Miescher found a phosphorus-containing substance in white blood cells unlike the proteins he expected to find. He called this substance a nuclein. Later, it became known as nucleic acid. Phoebus Levene’s Role in DNA’s Discovery: Fifty years later, in 1919, Russian-born biochemist Levene characterized nucleic acids as molecules made of phosphate, sugar, and four nitrogenous bases—adenine (A), guanine (G), cytosine (C), and thymine (T). Levene also differentiated the deoxyribonucleic acid (DNA) from the related genetic material ribonucleic acid (RNA). Oswald Avery, Colin MacLeod, and Maclyn McCarty Uncover DNA’s Genetic Role: By 1944, Avery, MacLeod, and McCarty, at the Rockefeller Institute in New York, showed that DNA—not proteins, as others had previously assumed—was the substance that passed along genetic information in experiments involving bacterial transformation. 2-36  Rosalind Franklin, Maurice Wilkins, and Raymond Gosling on X-ray Diffraction: Studies of DNA's structure through X-ray diffraction, by Maurice Wilkins and Raymond Gosling, began in 1946. Rosalind Franklin’s expertise was then brought to the team, where she contributed pivotal images of crystallized DNA and crystallographic calculations. These images were critical to solving the mystery of DNA's double helix structure. Erwin Chargaff and DNA’s Chemical Rules: In 1950, Chargaff published a paper outlining the chemical rules for how nucleotide bases pair—that adenine always pairs with thymine, and cytosine pairs with guanine. These paired bases are part of the DNA structure. James Watson, Francis Crick, and DNA’s Double Helix Structure: Watson and Crick's quest to discover the structure of DNA began with their first meeting in the summer of 1951. The2-37 model they initially proposed was wrong, featuring three strands  A big breakthrough came when, without Rosalind Franklin's knowledge, Watson and Crick gained access to her research—a report written for the Medical Research Council on the structure of DNA, obtained by Crick via his thesis advisor Max Perutz. Watson and Crick also got access to Franklin's X-ray diffraction images of crystallized DNA. The most notable was perhaps the image known as "Photo 51," which Franklin's graduate James Watson and Francis Crick student publishedRaymond Gosling their findings onhanded the double helix in a paper titledover to Maurice "A Structure forWilkins. Deoxyribose Nucleic Acid" in April of 1953. Though Franklin's work proved key to helping Watson and Crick devise their model, their paper included a mere footnote acknowledging that they were "stimulated by a knowledge of the general nature" of the unpublished work and ideas of Dr. M. H. F. Wilkins and Dr. R. E. Franklin and their King's College colleagues. 2-38  DNA Double Helix Watson & Crick model. DNA molecule composed of two chains of nucleotides. Two chains spiral around each other to form a pair of right-handed helices. Two chains are anti- parallel (see arrow). Sugar and phosphate backbone located outside of the molecule.  DNA Double Helix Bases situated inside the double helix, stacked perpendicular to the long axis. Two DNA chains held together by hydrogen bonds between each base on one chain and a base on the other chain. Double helix is 20 Å (2 nm wide). Pyrimidines are always paired with purines.  DNA Double Helix Major groove and minor groove. Double helix taking a turn every 10 base pairs (residues) (3.4 nm). Two chains being complementary to each other.  Summary The DNA molecule is a double helix, with sugar-phosphate backbones on the outside and base pairs on the inside. The bases pair in a specific way: – Adenine (A) with thymine (T) – Guanine (G) with cytosine (C) Importance of the Watson-Crick Model: Accounting for its functions. 1. Storage of genetic information - Containing records of instructions that determine all the inheritable characteristics that an organism exhibits. 2. Replication and duplication - Information for synthesis of new DNA strands. 3. Expression of the genetic message - Information in DNA must be used to direct the order by which specific amino acids are incorporated into a polypeptide chain. 2-46  Genes Made of RNA Hershey & Chase investigated bacteriophage, virus particle by itself, a package of genes – This has no metabolic activity of its own – When virus infects a host cell, the cell begins to make viral proteins – Viral genes are replicated and newly made genes with viral protein assemble into virus particles Some viruses contain DNA genes, but some viruses have RNA genes, either double- or single-stranded NMF/Cell/Intro 48  Physical Chemistry of Nucleic Acids DNA and RNA molecules can appear in several different structural variants – Changes in relative humidity will cause variation in DNA molecular structure – The twist of the DNA molecule is normally shown to be right-handed, but left-handed DNA was identified in 1979  A Variety of DNA Structures High humidity DNA is called When wound in a left- the B-form. handed helix, DNA is - base pairs perpendicular to termed Z-DNA the helix. One gene requires Z- Lower humidity from cellular DNA for activation conditions to about 75% and DNA takes on the A-form – Plane of base pairs in A- form is no longer perpendicular to the helical axis – A-form seen when hybridize one DNA with one RNA strand in solution A form B form Z form  Variation in DNA between Organisms Ratios of G to C and A to T are fixed in any specific organism The total percentage of G + C varies over a range to 22 to 73% Such differences are reflected in differences in physical properties  Chargaff’s rules of DNA base composition A=T G=C A+T ≠ G +C QUESTION: A DNA sample contains 21% adenine. What is the complete percentage base composition?  Answer... According to Chargaff’s rule, A=T, G=C; If A=21%, then T=21%, Total A + T = 42%. Therefore, since G+C = 58%, then G=C=29%  DNA Sizes DNA size is expressed in 3 different ways: – Number of base pairs – Molecular weight – 660 is molecular weight of 1 base pair – Length – 33.2 Å per helical turn of 10.4 base pairs Measure DNA size either using electron microscopy or gel electrophoresis  DNAs of Various Sizes and Shapes Phage DNA is typically circular Some DNA will be linear Supercoiled DNA coils or wraps around itself like a twisted rubber band  Supercoiled DNA (phage DNA) (a) Relaxed. (b) Supercoiled. (c) Gel electrophoresis Highly compact, supercoiled form moves much more rapidly than the relaxed form.  DNA Supercoiling Not restricted to small circular DNAs but also in linear, eukaryotic DNA. - allows for chromosomal DNA to be compacted, to fit into microscopic cell nucleus. - facilitating replication (DNA synthesis) and transcription (RNA synthesis). Topoisomerases catalyze the interconversion between relaxed and supercoiled forms of DNA. Relationship between DNA Size and Genetic Capacity How does one know how many genes are in a particular piece of DNA? – Can’t determine from DNA size alone – Factors include: How DNA is devoted to genes? What is the space between genes? – Can estimate the upper limit of number genes a piece of DNA can hold DNA Size and Genetic Capacity How many genes are in a piece of DNA? – Start with basic assumptions Gene encodes protein Protein is about 40,000 D – How many amino acids does this represent? Average mass of an amino acid is about 110 D Average protein – 40,000 / 110 = 364 amino acids Each amino acid = 3 DNA base pairs 364 amino acids requires 1092 base pairs  DNA Genetic Capacity How large is an average piece of DNA? – E. coli chromosome 4.6 x 106 bp ~4200 proteins – Phage (infects E. coli) 4.85 x 104 bp ~44 proteins – Phage x(one of smallest) 5375 bp ~5 proteins  Summary Natural DNAs come in sizes ranging from several kilobases (kb) to thousands of megabases (Mb) The size of a small DNA can be estimated by electron microscopy This technique can also reveal whether a DNA is circular or linear and whether it is supercoiled  The DNA of the chromosomes located within the nucleus Flow of Information in an contains the entire store Eukaryotic Cell of genetic information. Selected sites on the DNA are transcribed into pre-mRNAs (step 1) , which are processed into messenger RNAs (step 2). The messenger RNAs are transported out of the nucleus (step 3) into the cytoplasm, where they are translated into polypeptides by ribosomes that move along the mRNA (step 4). Following translation, the polypeptide folds to assume its native conformation (step 5).