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The Central Dogma.docx

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York University
BIOL 2900
Motti Anafi

The Central Dogma 9/17/2013 1:02:00 PM Flow of Information in a Cell:
 DNA (information storage) ->Transcription -> RNA (information carrier) -> Translatio900n (by ribosomes) -> Proteins (polypeptide chain) -> Phenotype (physical features of the genotype)
 * proteins lead to and make up the phenotype
 Phenotype: The observable physical or biochemical characteristics of an organism, as determined by both genetic makeup and environmental influences. Atom: most basic piece of matter (+proton, neutron and surrounded by - electrons); smallest chemical unit of matter Molecule: larger unit comprises of two or more atoms; atoms connected together by chemical bonds DNA: molecule responsible for transmission of information from one generation to the next in all forms of cellular life on this planet (mainly comprises of nitrogen bases) The DNA structure contains linkages between sugars, phosphates and nitrogen bases. Genome: complete set of genes Nucleotides:
 i) phosphate group
 ii) sugar (deoxyribose in DNA & ribose in RNA)
 iii) nitrogenous base
 *nucleotides are monomers Nucleic Acid Structure- Nucleic acids are made up of nucleotides which are the building blocks of DNA and RNA. The nucleotides are linked by covalent bonds between the phosphate of one nucleotide and the sugar of the next. Polymerization results in a linear spine which is composed of alternating sugars and phosphates, with bases extending from them (Bauman, pp.49). Phosphodiester Bond- Nucleotides polymerize to produce nucleic acids through formation of a phosphodiester linkage between phosphate group on the 5' carbon of one nucleotide and the OH- group on the 3' carbon of another since phosphate binds with oxygen in a hydroxyl group. - DNA has many nucleotides connected to each other by phosophodiester bonds. DNA Structure: DNA primary structure consist of sequence of nitrogen containing base which includes information in the form of molecular code. Deoxyribose – sugar back bone Phosphate group Nitrogenous Base: Cytosine (Pyrimidine) Thymine (Pyrimidine) Guanine (Purine) Adenine (Purine) *purine are double ringed molecules *pyrimidines are single ringed molecules - DNA are polymers of nucleotides consisting of a pentose sugar and a nitrogenous base which is bound to a phosphate. - In DNA base pairings consist of adenine and thymine, and guanine and cytosine - In RNA base pairings consist of adenine and uracil, and guanine and cytosine - There are 3 billion nitrogenous bases in human DNA and more than 99 % of these bases are same in all human beings. Two strands of DNA run in opposite directions held together by complementary base pairs with hydrogen bonds twisted into a double helix (Archaeal DNA is separated and organized with histones, chromosomal DNA is folded into loops) - Complement base pairs : A-T, G-C through hydrogen bonds. A-T are held together by two hydrogen bonds. C-G are held together by three hydrogen bonds. - base is the information in the form of a molecular code (on the inside/core) - The backbone is the phosphate and sugars (outside) this creates a negative charge and polar - Two grooves between backbones are called the major and minor groove. Most protein data is made in the major groove because the minor groove is too narrow - Bases are planar and perpendicular to the path of the backbone purine bases are composed of two rings (A and G) and pyrimidine bases are composed of one ring (C, T, U). Base stacking contributes to the stability of the double helix - The complementary base pairing is important for DNA replication and transcription - Moving toward medicine where DNA can be sequenced Chargaff discovered the pairing rules of DNA letters, noticing A=T, C=G DNA structure Summary: - DNA structure is made of two stands ranging in opposite direction. the two strands are antiparallel, one strand runs from 3' end to 5' end and the other from 5' to 3' end. -The two strands have complementary base pairing; if base pair on one strand are AATCGC then the base pair on other strand would be TTAGCG. The specificity of pairing of A with T and C with G makes the two DNA strands complementary. -The two strands are held together by base paring and they are also twisted into double helix. DNA Replication - this is also known as anabolic polymerization process -it requires monomers (building blocks) and energy (in the form of triphosphate deoxyribonucleotides) Initial Process involves: replication beginning at a nucleotide called the origin A cell removes chromosomal proteins, exposing the DNA helix DNA helix (enzyme) unzips the DNA by breaking the hydrogen and therefore leaving the bases as a replication fork Proteins then stabilize the strands to prevent them from rejoining DNA polymerase then binds to each cell and synthesizes DNA by adding nucleotides only to the hydroxyl group of the 3' end ** DNA polymerase replicates only from the 5' end to the 3' end DNA is antiparallel and therefore the replicated strands are synthesized in two ways: -Helicase is responsible for "unzipping" the DNA. Helicase breaks the loose hydrogen bonds. Free nucleotides floating in the cytoplasm are used to build new DNA, think of them like loose bricks. DNA polymerase are responsible for constructing DNA strands. Bauman, p. 203 Leading Strand- synthesizes continuously as a single long chain of nucleotides Lagging Strand- synthesizes in short segments that are later joined. For a simpler understanding on DNA, please visit this link by Khan Academy: selection/v/dna DNA Roles: Duplication/Replication:molecule replication and transmission in each cell division. It's role is to replicate and transmit itself in each cellular division (almost always identical) Bauman textbook chapter 7 Histones (stabilization proteins) are removed from the DNA, DNA Helicase unwinds the DNA by breaking the hydrogen bonds between the complementary base pairs and essentially unzipping it (this forms the replication fork), DNA is always replicated in the 5' end to 3' end, therefore the leading strand (the strand which runs from 5' end to 3' end)synthesizes uninterrupted or continuously by the DNA polymerase. However, the lagging strand (complementary strand which runs from 3' end to 5' end) must be synthesized backwards and in fragments because of the direction it runs. It is not continuous, but synthesized in short segments (that are later joined) called Okazaki fragments. RNA Primase lays down RNA primers, DNA Polymerase III lays down new DNA in between the primers. A different polymerase (DNA polymerase I) replaces the RNA primers with DNA. Finally, DNA ligase fuses the fragments together. 
 *at; 95 degrees the original DNA strand denatures
 60 degrees primers finds its place
 72 degrees DNA polymerase makes another copy of DNA Information: information expression within molecule (sequence of nucleotides). DNA expresses its information by transcription to RNA (m RNA) which is translated into proteins by the ribosomes. Information is expressed into sequenced nucleotides. The sequences transcribed into RNA in order to release their information. DNA vs RNA DNA RNA Contains a Deoxyribose Contains a Ribose sugar. sugar. Consists of A,T,C,G Consists of A,U,C,G Double-Stranded Single-Stranded It's the genetic material of Is for protein synthesis in all cells and all cells and DNA virus genetic material of RNA viruses * DNA thymine (T) is replaced by uracil (U) in RNA * DNA Replication: DNA helix unwinds resulting in single DNA strand ; this is brought about by the enzyme Helicase. DNA polymerase attaches to DNA DNA polymerase brings the correct nucleotides and also checks for errors in the sequence before forming new DNA strands After copied polymerase falls off The cell divides, one set on DNA molecule goes to each cell DNA replication is semi-conservative meaning out of the two DNA strands in daughter cell one is the original strand from the parent cell and other is complementary replicated copy of the parent strand. DNA replication is also an anabolic polymerization process, and allows a cell to pass copies of its genome to its descendants. DNA Amplification: used for forensic analysis to identify pathogens. Creates multiple copies of the original DNA sequence. 
 Polymerase Chain Reaction (PCR): - Acts as a temperature regulating system.
 - Quick amplification to detect specific DNA sequence by altering temperature. - PCR uses: used to detect specific genes for genetic research. Bauman (2012) says that scientists used this method to study the genome of a previously unknown pathogen that killed people in 2003 with SARS. PCR helped scientist determine the nucleotide sequence, which was found to be similar to coronaviruses. -PCR can also be a tool used for genetic mapping: provides information on organism's metabolism and growth; locating genes; nucleotide sequencing of pathogens. Essentially, PCR is used to create multiple copies of the DNA molecule, which is used for in various applications for genetic research (Bauman, 2012). o - Denaturation: Alters temperature to 95 C, in which the heat denatures DNA, breaking the hydrogen bonds that hold the strands together, exposing the bases (no need for helicase). The DNA strand is now open to be replicated. - Priming:Temperature is then reduced to 60 C so primers can anneal (form hydrogen bonds) with complementary sequences. 
 - Extension: Then alters temperature to 72 C o (allows for optimal functioning of DNA polymerase) to allow the enzyme DNA polymerase to copy in both directions and attach nitrogen bases with the correct pairing to form new DNA strand.
 - These 3 temperature changes repeat to amplify protein (i.e. cycle 3 has 8 copies, cycle 4 has 16 etc.) By changing the temperature from to 95 then to 60 and to 72, the DNA polymerase keeps duplicating the DNA. - PCR is the DNA duplication that is done in a tube. - What do we need for PCR? We need primers, DNA polymerase, nucleotides. Transcription: (DNA -> mRNA) the enzyme complex called RNA polymerase causes the DNA strands to separate over a short region (10-20 base pairs). The polymerase move along the DNA, and as it does, it forms an RNA chain using free nucleotides. The order of the nucleotides in RNA is determined by the order of the nucleotides in one of the DNA strands through the complementary base-pairing rule. RNA is shorter than DNA and it contains the base uracil (U) instead of Thymine (T). If the first letter RNA polymerase encounters in DNA is a T, the enzyme will add an A to the chain and if the next DNA letter is G, a C will be added to the new chain. Translation: (mRNA -> polypeptide chain) ribosomes use the mRNA sequences to build polypeptides with the help of transfer RNA (tRNA) which is responsible for identifying the correct sequence of CODON (group of 3 nucleotides) so as to present complimentary amino acid. Each ribosome contains 3 sites for tRNA to bind (A, P, and E). A site is where tRNA initially binds, tRNA in the P site results in the amino acid binding to the growing polypeptide chain, E site releases tRNA) DNA (double stranded): Is a double stranded molecule and it contains thousands of hydrogen molecules between the bases making it a weak bond, but at a normal temperature they form a stable double stranded DNA molecule. DNA carries instructions (information) for the synthesis of RNA and proteins. Cells replicate their DNA molecules and pass the copies on ensuring they are viable for life. DNA is self replicating Is made up of a deoxyribose sugar, phosphate backbone and complimentary bases of Adenine-Thymine (A-T) and Cytosine-Guanine (C-T) RNA (usually single stranded): Acts as an enzyme and binds amino acids together to form polypeptides. A monomer of nucleic acids is a nucleotide. RNA is synthesized from DNA when needed. RNA is made up of ribose, phosphate backbone and complimentary bases of Adenine-Uracil (A-U) and Cytosine-Guanine (C-T) Genotype: Is the actual set of genes in its genome. Phenotype: refers to the physical features and functional traits of an organism (i.e. structures, morphology and metabolism). In any circumstance genotype determines phenotype by specifying what kinds of RNA and structural, enzymatic and regulatory protein molecules are produced. The transfer of genetic information: First: make an RNA copy of the gene = Transcription This information is copied as RNA nucleotide sequences... RNA molecules synthesize polypeptides = Translation These processes make up the Central Dogma of genetics: DNA is transcribed to RNA, which is translated to form polypeptides. (Analogy p. 206 Bauman). The flow of genetic information in cells is therefore from DNA to RNA to protein. All cells, from single-celled bacteria to humans, express their genetic information in this way - this principle is so fundamental that it is termed the Central Dogma of molecular biology (Alberts, Johnson, Lewis et al., Molecular Biology of the Cell, 4th ed., pp. 301). Cells transcribe four main types of RNA from DNA: RNA primer- needed for DNA replication. mRNA- carries genetic information from chromosomes to ribosomes. rRNA- combine with polypeptides to form ribosomes. tRNA-deliver the correct amount of amino acids to ribosomes based upon the sequence of neucleotides. Events in transcription (3): Initiation: RNA polymerases (the enzyme that synthesizes RNA) bind to specific DNA molecules called promoters. In bacteria Sigma factor is necessary for recognition of a promoter. RNA polymerase (once adhered to a promoter) unzips and unwinds the DNA molecule. It forms a bubble. Elongation: RNA transcription being 10 spots away from the promoter. RNA polymerase links together DNA and RNA, synthesizing RNA. The enzyme moves down the DNA strand, elongating the RNA strand. No primer needed. Triphosphate ribonucleotides is the energy source for the transcription process. Termination: Complicated. In bacteria there are two ways: Self termination: Occurring when RNA polymerase transcribes a terminator sequence of DNA is composed of two symmetrical series: 1 rich in guanine and Cytosine and the other rich in adenine. When RNA polymerase transcribes the adenine rich portion of the terminator, DNA and the uracil bases of RNA cannot withstand the tension, and the RNA transcript breaks away form the DNA, releasing RNA polymerase. Rho-Dependent Termination: Rho, binds to a specific RNA sequence near the end of an RNA transcript. Rho protein moves toward RNA polymerase at the 3' end of the growing RNA molecule, pushing between RNA polymerase and the DNA stand... forcing them apart. Microbiology week 2 9/17/2013 1:02:00 PM Proteins Amino acids are the building blocks of proteins Proteins are made by the grouping of amino acids The DNA and RNA help to make specific proteins Translation  Process of converting info stored in nucleic acid sequences into proteins  Genetic code: the ribosomes read mRNA sequences in 3-base codons Components of Translation mRNA – the template is used to specify acid pattern Ribosomes – a complex of proteins and rRNA molecules Transfer RNA – small RNA molecules serve as adaptors between codons in mRNA and amino acids. Transfer RNA anticodon recognize the codon in the mRNA Genetic Code Universal code used in the nuclear genome Complimentary anticodons are present in tRNA molecules, specifically linked to amino acids With 4 bases in RNA and 3 base codons, there are 64 posible codons but only 20 amino acids. * Uracil – RNA instead of DNA (Thymine)* Polyribosomes Strings of ribosomes, assembled along an mRNA to increase rate of protein production Ribosomes translate the MRNA (many at a time) Ribosome reaches the stop protein and detaches from the protein molecule Recombination of DNA DNA of interest Bacterial genome Section of DNA breaks/cut there are 2 sticky ends that meet Mutations  In the living cell, DNA undergoes frequent chemical changes, when it is being replicated  Most of these changes are quickly repaired  Those that are not result in a mutation  Thus, mutation is a failure of DNA repair Molecular base of mutation A mutation is any change in the organisms DNA DNA mutations affect phenotype only when the mutation expresses (DNA – RNA – protein) and the resulting protein functions abnormally. Not all mutations affect the proteins function Most common is a point mutation (change in a single nucleotide) Non complimentary nucleotide is paired. (Change in the second triplet) Insertions and deletions result in a shift change – drastically changing the protein Plasma Membrane  Every cell  Function: separates the inside of a cell from the outside environment  Separate cells from one another  Provide a surface chemical reactions occur  Regulate the passage of material in and out of the cells (looks like a plastic bag) Membrane structure Structure/Function of Bacteria 2013-09-25 3:58 PM 9/17/2013 1:02:00 PM Prokaryotes Vs. Eukaryotes All cell life have the same characteristics: Cell membrane (regulates flow of nutrients and wastes that enter & leave cell) DNA as its genetic material RNA molecules Proteins Enzymes Basic chemicals (carbs, fats, vitamins etc.) Reproduction Energy Prokaryotic Cell Structure (Inside – Out) flagellum (tail – allows bacteria to move - way to sense chemicals) – also known as a little motor for movement, similar to a propeller, can change direction and speeds. Structured like a tail(s). Function – rotation propels bacteria through the environment. Used in chemo taxis – movement toward/away from substances based on gradient of concentration. Axial Filaments: spirochete: cause leprosy Similar to flagella Snake like movement Runs lengthwise along cell Fimbria and Pilli helps bacteria stay in the same place. Sticky projections used to adhere one to another to hosts, and to environmental substances Fimbriae can be distributed evenly or over the entire surface May be hundreds per cell and are shorter than flagella Pilli Long hollow tubules Longer than fimbriae Only one or two per cell Join bacteria and mediate transfer of one cell to another (conjunction) Capsule & Slime layer structure Sticky substances surrounding cell Almost always observed on the surface Composed of polysaccharides, polypeptides, or glycoproteins Mediates adherence of cells Protect bacterial cells from engulfment Prevents cells from drying Reserves of carbs Biofilm Complex aggregation of microorganisms growing on a solid substrate Mediated by fimbria & capsules More resistant than planktonic cells to antimicrobial agents, and host immune responses In medicine, biofilms spreading along implanted tubes or wires can lead to severe infections in patients Cell Wall Allows bacteria to resist osmotic stress Prokaryotic cell wall – provides structure & shape Assists in some cells attaching to other cells Not found in eukaryotic animal cells We can target the cell wall of bacteria with antibiotics Peptidoglycan Single macromolecule Highly cross-linked Surrounds cell Provides rigidity Bacterial Shape Structure – chain, alternating sugar (NAM, NAG, NAM, NAG) Cell Wall Made of Peptidoglycan Cell wall structure based on 2 sugars N-acytl glucosamine (NAG) N-acytl muramic acid Gram Positive & Gram negative Bacteria 9/17/2013 1:02:00 PM Gram Positive & Gram negative Bacteria The cell walls of certain bacteria (denoted Gram-positive) retain the first dye and appear violet, while those that lose it (denoted Gram-negative) appear red. Also called Gram's method. Periplasmic Space The periplasm is a space bordered by two selective permeable barriers, i.e., biological membranes, which are the inner membrane (i.e., cytoplasmic membrane) and the outer membrane in Gram-negative bacteria. There is no periplasmic space in Gram-positive bacteria because there is only one biological membrane, the cytoplasmic membrane. A region termed "inner wall zone" (IWZ) has been observed between the cytoplasmic membrane and the mature cell wall. Toxins Chemicals produced by the pathogen Harm tissues or trigger host immune responses that cause damage: Exotoxins (a toxin released by a living bacterial cell into its surroundings.) Endotoxins (a toxin that is present inside a bacterial cell and is released when the cell disintegrates. It is sometimes responsible for the characteristic symptoms of a disease). Exotoxins: - Proteins secreted by live pathogen abnormal signal to cells destroy cellular and extracellular structures Necrotizing Fasciitis * * Pathogens are developing resistance to multiple drugs, some to nearly all. (MRSA). * Endotoxins: Gram negative contains LPS (endotoxin) LPS (Lipopolysaccharide) LPS structure: O-side chain, Core polysaccharide, Lipid A O-chain: chain varies depending on bacterium Core-Polysaccharide: relatively constant composition Lipid A: composed of glycolipids associated with toxic activity in gram negative bacterium Lipopolysaccharide (LPS) May play a role in infection by any gram negative bacteria Released from dead cells when cell wall disintegrates Causing inflammation Activates macrophages, neutrophils Produce pyrogens (may trigger fever) Vasodilation LPS as an endotoxin Acute Inflammation Release of too much LPS Big problem! Can be induced by antimicrobial drugs that kill bacteria Systemic infection (sepsis) Activates blood coagulation Acute whole body inflammation Drop of BP Shock Prokaryotic cell Organization and Structure The Cytoplasm the material or protoplasm within a living cell, excluding the nucleus. Prokaryotic chromosome Typically one large circular molecule of DNA With no nuclear membrane May have plasmids (a genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular DNA strand in the cytoplasm of a bacterium or protozoan.) Main roles of bacterial DNA Contain the genetic material Transcription replication Ribosomes a minute particle consisting of RNA and associated proteins, found in large numbers in the cytoplasm of living cells. They bind messenger RNA and transfer RNA to synthesize polypeptides and proteins. Protein synthesis Some differences from eukaryotes The ribosomes of prokaryotes (70S) are smaller then cytoplasmic ribosomes of eukaryotes (80S) But 70S ribosomes can be found in eukaryotes Endospores A resistant asexual spore that develops inside some bacteria cells. the inner layer of the membrane or wall of some spores and pollen grains. Formed by a few groups of gram positive bacteria as intracellular structures but ultimately they are released as free spores Formed by vegetative cells in response to environmental signals that indicate a limiting factor for vegetative growth E.g. exhaustion of an essential nutrient Endospores exhibit no sings of life. Although, they retain viability indefinitely Mature spores are Highly resistant to environmental stress such as: High temperature, irradiation, strong acids, disinfectants etc. Endospores germinate and become vegetative cells when the environment stress is relieved Endospore – formation is a mechanism of survival rather than a mechanism of reproduction Genetic Changes in Bacteria Mutations in Bacteria Mutations arise in bacterial populations Spontaneous Induced Cancer treatment Radiation Some chemotherapy agent Rare Mutations are expressed as a phenotype however, bacteria are haploid. ((of a cell or nucleus) having a single set of unpaired chromosomes. Compare with diploid. (of an organism or part) composed of haploid cells) Rapid growth rate Selective advantage enriches for mutants Exchange of Genetic Information in Bacteria Horizontal gene transfer Donor to Recipient (unidirectional) – Transfer only Donor does not give an entire chromosome Gene transfer can occur between species Three Types Transformation DNA from dead bacteria Transduction By a bacteriophage Transformation Naturally competent bacteria are able to take up exogenous DNA and undergo genetic transformation Transduction Gene transfer from a donor to a recipient by way of a bacteriophage. Bacteriophage structure Head (capsid) – genetic material is here Nucleic acid Protein Protection Infection Infection of Host Cells by Phages Absorption Tail fibers Receptor is LPS for T4 Irreversible attachment Base plate Sheath contraction Generalized Transduction Potentially any donor gene can be transferred Infection of Donor Phage replication and degradation of host DNA Assembly of phages particles Release of phage Infection of recipient Recombination into host DNA Conjugation Gene transfer from a donor to a recipient by direct physical contact between cells Mating types in bacteria Donor F-factor (Fertility factor) Pili Clinical Significance of Transformation Conjugation and Transduction Rapid spread of harmful bacteria genes Virulent factors E. coli Antibiotics resistance Sterilization All killed Non-selective Autoclaving 121 degree c (heat/pressure) Heat resistant materials Ethylene oxide Non-heat resistant materials Usually equipment Very toxic Ultra violet light E.g. surfaces (operating rooms) Not totally effective Gamma radiation Food Disinfection Liquids that kill bacteria E.g. Phenol based Too toxic for the skin surfaces Antiseptics Usually skin Eg. Iodine or 70% alcohol or isopropanol Reduce bacterial load Antibacterial Agents Natural “antibiotics” (primitive medicine – e.g. Honey for cough?) Herbal remedies Chicken soup cures a cold? What are Antibiotics Antibiotics are any natural substances secreted by one microorganism against another microorganisms. Bactericidal antibiotics Irreversible drug. The effected microbes are dead Bacteriostatic Reversible growth inhibition Treatment of both is sufficient if host defenses mechanisms can take care of the residual cells Antibiotics work together with the immune system Antibiotics Selective drugs No (or limited) harm to patient Destroy structures/ stop functions present in bacteria Not present in host Diversity of Prokaryotes 9/17/2013 1:02:00 PM Diversity of Prokaryotes Rod-like (Bacillus) Spiral (Spirillum) Spherical (coccus) Environmental Conditions Temperature Grows best below 20 degrees C (psychrophiles) Grows best between 20 – 50 Degrees C (mesophiles) Grows best above 50 degrees C (thermophiles) pH (Acidic or Basic) Environments Grows well at pH of 1 to 2 (Acidic) (Acidophiles) Grows best near neutral pH (Neutrophile) Grow well at pH as high as 9 (basic) (Alkaliphile) Water Most bacteria require a minimum moisture Spores: near absence of moisture Salt Most bacteria require a moderate level of salt Some cells can exist in very high salt concentrations (halophiles) Oxygen Availability Require O2 for growth (aerobic) Require lack of O2 for growt
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