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Chapter 10 – Molecular Biology of the Gene The structure of the Genetic Material I. Intro A. Viruses – basically packaged nucleic acid particles 1. Living or Non-living a) genetic material is nucleic acid, (1) not cellular (2) does not reproduce on its own. b) Nucleic acid wrapped in a protein coat (capsid) and c) capsid and nucleic acid is sometimes surrounded by a membranous envelope (phospholipids + membrane proteins) d) They are tiny (largest are 200nm – 1/100th of a human cell) and (1) do not carry the tools they need to reproduce (2) they need the cell’s tools! 2. Brief life cycle of Herpevirus (similar to other viruses) a) Viruses Gain access to specific cells (nerve cells in this case) (1) trick it - proteins on virus (the ligand) fit into and bind with natural cell receptors – structure function. (2) cell takes up virus, unknowingly b) viral DNA enters the nucleus c) It can remain dormant until the time is right d) Then hijack the cell and use the cellular machinery for its own purpose (to reproduce of course…). e) Cell fills with virus particles and lyses, releasing the particles to infect other cells (the sores). 3. Once infected, it remains permanently latent, integrated into the nerve cell’s DNA. 4. 75% Americans carry HSV-1 and 20% have HSV-2 5. many infected people never show symptoms 6. ability to remain latent is somewhat unusual, a trait shared with HIV 7. Viruses are far simpler on the molecular level compared to Mendel’s peas and Morgan’s flies a) because of this, virus gave us our first glimpse of how DNA controls heredity – molecular biology B. The chromosomal theory of inheritance set the stage for the development of a molecular understanding of the gene C. Remember, we still don’t know what molecule(s) is responsible for heredity. We know it has to do with chromosomes, and we know it is made of protein and DNA. So which is it? Scientists thought protein. II. How was DNA determined to be the genetic (hereditary) material of life? A. Began in 1928 – English bacteriologist Frederick Griffith 1. He heat-killed a pneumonia-causing bacteria Streptococcus pneumonia (smooth strain) 2. Took some substance from the dead bacteria and gave it to a harmless form of the bacteria (rough strain) 3. The rough strain and its descendents were transformed into the infectious smooth strain! 4. What was this “transforming factor”? Griffith and everyone else believed it to be protein! 5. Inject: Rough strain Smooth strain Heat-killed smooth strain Rough strain + heat-killed smooth strain Outcome: Mouse lives Mouse dies Mouse lives Mouse dies B. Chromosomes were known to be composed of: 1. Protein – more complex – 20 amino acids – therefore they believed it to be the genetic material 2. DNA C. Oswald Avery continued Griffith’s experiments 1. separated the different components of the heat-killed smooth Streptococcus pneumonia into the 4 major macromolecules: polysaccharides, proteins, nucleic acids, and lipids. 2. mixed each component separately with the rough Streptococcus pneumonia and injected each into mice. 3. Nucleic acid was the lethal mixture 4. Inject: Rough strain + lipids Rough strain + carbs Rough strain + proteins Rough strain + nucleic acid Outcome: Mouse lives Mouse lives Mouse lives Mouse dies!! 5. So is it DNA or RNA? 6. He treated the nucleic acid with an enzyme that hydrolyzes RNA (RNase), added it to the Rough Streptococcus pneumonia and it killed the mice…DNA was it!! Inject: Rough strain + nucleic acid + RNase Outcome: Mouse dies!! D. Hershey and Chase experiment – 1952 – confirmed DNA to be hereditary material. 1. Studied T2 bacteriophages a) bacteriophages (bacteria eaters) are bacterial viruses – made of protein and DNA 2. They knew the T2 could take over bacterial cells, they wanted to confirm that is was DNA doing this and not protein. 3. The experiment (Fig. 10.1AB): a) Grew phage in different radioactive elements b) radioactive Sufur to label protein (S only in the proteins), radioactive phosphorous to label DNA (phosphorus only found in the DNA). c) use phage to infect bacteria (E. coli) d) blend to dislodge any phage material stuck to outside of cell e) centrifuge cells down and look for radioactivity in the cells f) phage with labeled DNA resulted in radioactive cells. Thus, DNA was being injected into the bacteria. g) return bacteria to growth media (liquid that bacteria grow in) and they lyse, releasing virus with radioactive DNA, but not protein. 4. Conclusions – a) only DNA is injected, protein left outside b) it is the injected DNA that cause the cells to produce additional phage 5. These results plus others gathered during the 1920’s through the early 1950’s convinced the world that DNA was the hereditary material. 6. This set the stage for one of the most controversial and celebrated quests in all of science, the structure of DNA! III. DNA and RNA are polymers of nucleotides A. By 1952, scientists knew a great deal about DNA including all of its different atoms types and how they are covalently bonded to each other. B. What they did not know was the specific three dimensional arrangement of these atoms that gave DNA its unique properties 1. the ability to store genetic information 2. copy it 3. pass it to the next generation C. nucleotides – monomers of nucleic acid – phosphate, sugar, nitrogenous base D. polynucleotide – polymer of nucleotides E. sugar phosphate backbone - Nucleotides attach to each other via the phosphate of one nucleotide and the sugar of the next F. DNA – DeoxyriboNucleic Acid 1. Deoxyribo- deoxyribose sugar 2. Nucleic – found in the nucleus 3. Acid – acidic phosphate group G. 4 flavors of bases found in DNA – 2 groups 1. Purines (double-ring structure) a) Adenine (A) b) Guanine (G) 2. Pyrimidines (single ring structure) a) Cytosine (C) b) Thymine (T) H. RNA – ribonucleic acid 1. Ribose sugar instead of deoxyribose (compare sugar structures) 2. Phosphate is the same as DNA 3. RNA uses the same nitrogenous bases as DNA except instead of Thymine (T) it uses Uracil (U) – show how similar 4. Summary : Ribose instead of deoxyribose, U instead of T, single stranded vs. double stranded I. DNA is a double helix 1. Structural denotes function (hammer for nails) – so learning the shape of DNA was critical to understanding its role in heredity 2. The race for the structure of DNA was on! 3. The “winners”: James D. Watson (American) and Francis Crick (English). a) Science is not a solitary act – they had a great deal of information like the structure of nucleotides, Rosalind Franklin’s X-ray crystallographic pictures of DNA – used to deduce the helical nature, Erwin Chargaff’s data showing that the amount of A and T, and C and G were always equal, and previous knowledge that different species had different ratios of A + T to G + C. – mention the book 4. The model that fit all the data was a double helix (a twisted rope latter) with sugar-phosphate backbones as the rope rails and the nitrogenous bases as the steps. 5. A always bonds with T and C always bonds with G – called base pairs 6. No restriction on linear sequence of nucleotides 7. Structure was published in 1953 and led immediately to the proposed mechanisms about DNA function. 8. James D. Watson was the Cold Spring Harbor Laboratory Chancellor here on Long Island until October 14th 2007 when suspended from his responsibilities due to comments he made that were published in the The Sunday Times (U.K.). DNA REPLICATION IV. DNA replication depends on specific base pairing A. Complete and faithful copies of DNA must be produced (replicated) during the cell cycle B. Watson and Crick proposed a model for how DNA replicates (is copied) C. The mechanism proposed and confirmed at the end of the 1950’s = semi-conservative model 1. each polynucleotide strand acts as a template on which a new strand can be assembled according to base pair rules (A-T, C-G). D. Although it sounds simple, many proteins are involved. 1. DNA needs to be unwound, held open, and the proper bases must be inserted one at a time by enzymes called DNA polymerases over millions and millions of bases!! E. The copy mechanism is VERY precise at a speed of 50 to 500 per second. There is a mistake on average of 1/1,000,000,000! V. DNA replication: A closer look A. ORIGINS OF REPLICATION (ORI) 1. many occur at same time in eukaryotes (speeds up replication) 2. Replication bubble - forms at ORI – DNA is unwound and unzipped. 3. replication goes in both directions B. DNA polymerases – enzyme (protein) that synthesizes DNA 1. Reminder: DNA is polar (different ends - there is a 3’ end and 5’ end) 2. DNA polymerase can only attach nucleotides to the 3’ end of a growing daughter strand 3. Therefore, replication proceeds 5’ to 3’ a) Leading strand – made continuously 5’ to 3’ b) Lagging Strand – made in short pieces (Okazaki fragments) - “glued” together by DNA Ligase. 4. DNA polymerases can also proofread the new daughter strands to see if they made a mistake C. More Workers: 1. DNA helicase – enzyme that unzips the DNA 2. Why do the DNA strands stay separated and not just zip back together? Single stranded binding proteins coat the swingle strands and don’t allow it to. 3. DNA pol cannot start from scratch, it always needs an RNA primer that is added by the enzyme Primase 4. Now there is RNA in the DNA? Don’t worry, primer is replaced by DNA later by another type of DNA polymerase (there are many types) D. DNA replication ensures that the genetic information is accurately copied and passed along to the daughter cells VI. THE FLOW OF GENETIC INFORMATION FROM DNA TO RNA TO PROTEIN A. DNA = molecule basis of genotype B. proteins = molecular basis for phenotype C. 1940’s American geneticists George Beadle and Edward Tatum – studying nutritional mutants of the mold Neurospora. 1. Found that genetic mutants lacked single enzymes needed to complete metabolic pathways 2. one gene-one enzyme hypothesis 3. extended to the one gene-one protein hypothesis and later restricted to the one gene-one polypeptide hypothesis D. The flow of information in gene expression is from DNA to RNA (transcription) to polypeptide (translation). E. 80 to 90% of human DNA is referred to as “junk” DNA, meaning we have no idea what it is used for. Only 1-2% of our 3billion base pairs are genes!! VII. The Codon A. DNA = a parts list with the parts being the proteins B. Written using its own alphabet of only four letters A, T, G and C corresponding to the nucleotides. C. The list can be read just like a shopping list, we just needed to learn how to read it. D. Letters of the DNA alphabet form words, always only 3 letters long (triplet code) called codons E. Each codon corresponds to a specific amino acid. 1. How many amino acids are there? 2. Compare that to how many possible codons there are. 3. So why does DNA use 3 letter words and not 2 or 1? F. These words form sentences of varying length called genes of which there are 30,000+ in humans, 1000’s per chromosome G. Genes are stuck inside the nucleus, however, and protein needs to be made in the cytoplasm H. Transcribe a copy of the gene as a mRNA (it carries the message), which can cross the nuclear envelope and enter the cytoplasm. VIII. The genetic code is the Rosetta stone of life A. The Genetic code tells us how to decipher the nucleic acid language IX. Transcription - produces genetic messages in the form of RNA A. In prokaryotes, transcription and translation both occur in the cytoplasm. B. In eukaryotes,the transcribing of DNA to messenger RNA occurs in the nucleus C. It is similar to replication in that the two DNA strands are unwound and unzipped. D. Only one strand serves as the template E. RNA nucleotides follow the same base pair rules as DNA except that U pairs with A instead of T. F. RNA polymerase – the enzyme that transcribes the gene into mRNA by polymerizing the appropriate RNA nucleotides 1. Synthesizes mRNA 5’ to 3’ just like DNA polymerase G. 3 stages 1. Initiation – RNA polymerase binds to the promoter, DNA unwinds, RNA synthesis begins – promoter tells RNA polymerase which strand to transcribe 2. Elongation – RNA polymerase continues polymerizing RNA nucleotides along the DNA template according to base pairing rules. As the RNA strand is made, it peels away from the DNA as the DNA winds up again. 3. Termination – RNA polymerase reaches a special terminator sequence of DNA bases, detaches and mRNA is released. X. RNA processing A. Messenger RNA (mRNA) – carries the message from DNA to ribosome (translation machinery) outside the nucleus. B. The mRNA is processed BEFORE leaving the nucleus 1. Cap and tail are added to the mRNA a) Protects mRNA from cellular enzymes that would otherwise degrade it. b) Helps ribosome to recognize the mRNA 2. Non-coding regions are removed (RNA splicing) a) Most plant and animal genes have INTRONS and EXONS (1) Introns – non-coding regions that may be cut out (2) Exons – coding regions used to make protein (3) Introns are removed and exons are spiced together in a process known as RNA splicing. (4) Alternative splicing – splice different combinations of exons to get different mRNAs – so one gene actually can code for multiple polypeptides! XI. Translation A. Translation – rewording of a message into a new language (nucleic acid language amino acid language) 1. Transfer RNA (tRNA) a) the translator – converts nucleic acid language to amino acid language. b) How do cells make tRNA? There are tRNA genes – no translation of course c) Structure- Function of tRNAs (1) Single strand of RNA, ~80 nucleotides (2) Parts of a tRNA (a) Folds upon itself to form double-stranded regions (b) amino acid attachment site (c) single-stranded loop with special triplet of bases called an anti-codon. (i) Anti-codon – complementary to the mRNA codon 2. tRNA synthetase – enzyme that attaches the proper amino acid to the proper tRNA to make an aminoacyl tRNA a) Amino acids are readily available in the cytoplasm from recycled proteins, digested food, or biosynthetic pathways. B. Ribosomes 1. huge translation machine 2. Structure a) composed of protein and rRNA (ribosomal RNA) b) arranged in two massive subunits (1) small subunit (2) Large Subunit c) There are two tRNA binding sites (1) P site (peptidyl site) – binds the tRNA with the growing peptide (2) A site (aminoacyl site) – binds the tRNA with the next amino acid to be added (aminoacyl tRNA) 3. Function a) coordinates the mRNA, tRNA, and growing peptide chain to allow synthesis. C. Translation can be divided into 3 phases similar to transcription 1. Initiation a) small ribosomal subunit and appropriate tRNA (MET attached and UAC anitcodon) binds to the start (initiation) codon AUG b) Large ribosomal subunit attaches placing initiator tRNA in the P site. 2. elongation a) codon recognition – anticodon of incoming tRNAamino acid complex binds with the codon at the ribosome’s A site. b) peptide bond formation – a polypeptide bond is formed between the growing polypeptide (attached to tRNA in P-site) and the new amino acid and the entire chain is now on the A-site tRNA. (1) Formation of the peptide bond is catalyzed by an enzyme within the ribosome c) translocation – The P-site tRNA leaves, the ribosome slides over, the A-site tRNA-polypeptide chain complex is now in P-site. - repeat back to step 1 3. Termination a) elongation continues until a STOP CODON (UAA, UAG, UGA) is reached. b) release factor – a protein that binds stop codon in the A site signaling the end c) ribosome subunits fall off to find a new mRNA to translate. D. The polypeptide folds into its tertiary structure both during and after translation. E. After translation, a number of polypeptides may come together to form a protein with quaternary structure. XII. Review: The flow of genetic information in the cell is DNA RNA protein A. Transcription – synthesis of mRNA complementary to a DNA template – in the nucleus B. Translation – conversion of info within mRNA to a polypeptide – in the cytoplasm XIII. How can genes be altered? A. Mutation – any change in the nucleotide sequence of DNA B. Two general flavors of mutation 1. Substitutions (point mutations) – change one base to another a) 3 possible outcomes (1) may change the encoded amino acid resulting in an abnormal gene product: (a) Sickle Cell anemia – a single substitution in the gene for hemoglobin (AT) resulting in a change in the sixth amino acid from Glu to Val. (2) The change may do nothing at all: (a) If new codon still codes for the same amino acid (silent mutation) (b) If the change in amino acid does not affect the function of the protein (3) In rare cases, base substitutions lead to genes that may enhance the success of the individual. (a) Such mutations provide genetic variability that MIGHT lead to the evolution of the species 2. Deletions and Insertions a) Tend to be more severe – can result in a frame shift, affects all of the amino acids downstream (1) reading frame = the triplet grouping of codons b) Will almost always result in a non-functional polypeptide C. Mutagenesis (formation of a mutation) can occur: 1. Spontaneously - Error in DNA replication or recombination, or other mutations of unknown cause 2. Mutagens - physical (radiation) and chemical agents. D. Mutagenesis might result in cancer (mutate genes involved in halting the cell cycle – p53 – the “guardian angel gene”) 1. can activate DNA repair proteins when DNA has sustained damage. 2. It can hold the cell cycle at the G1 check point on DNA damage recognition (if it holds the cell here for long enough, the DNA repair proteins will have time to fix the damage and the cell will be allowed to continue the cell cycle.) 3. It can initiate apoptosis (programmed cell death) if the DNA damage is irreparable. E. While mutations are usually harmful, they are also extremely useful: 1. Responsible for the rich diversity of genes in the world, making evolution by natural selection possible 2. Essential tools for scientists – a) Mendels naturally occurring mutations or Morgans X-ray induced mutations generate different alleles for study b) Also important for understanding protein function – change one amino acid to another and see how the protein reacts VIRUSES: PACKAGED GENES A. Viruses are essentially nothing more than packaged genes B. All genes want is to reproduce themselves (otherwise they would not be around today). 1. Viruses depend on their host cells for replication, transcription, and translation just as computer viruses depend on your computer. XIV. Bacterial phages (viruses) 1. reproduce in two general ways: a) Lytic cycle – phage invades host cell, hijacks it like a bank robber with a list of demands! It immediately tells the host to replicate the viral nucleic acid, transcribe and translate its protein-coding genes, assemble new viruses, and causes the host cell to lyse (pop), releasing the new reproduced phages. b) Lysogenic cycle – the phage DNA quietly enters the cell and inserts sneakily into the host DNA by recombination and becomes a prophage (the bank robber gets a job at the bank first). (1) When the host replicates, so does the viral DNA – can go on for MANY generations (2) Environmental cue will direct the prophage to switch to the lytic cycle (hold up the bank). XV. Animals Viruses and Disease 1. Organisms from all 5 kingdoms (3 domains) have viruses that infect their cells 2. Viruses have evolved to use DNA or RNA as their genetic material 3. Can be enveloped or not enveloped 4. Go over viral structure – envelope, glycoprotein spikes, protein coated RNA B. Enveloped RNA virus – Influenza, mumps (reproduce outside the nucleus) 1. Go over reproductive cycle - Figure 10.18B a) Envelope fuses and protein-coated RNA enters cytoplasm b) Enzymes remove protein coat c) RNA strand is used as a template to make complementary RNA strands (1) Serve as mRNA for protein synthesis (2) Serve as template to make new viral genome d) New coat proteins assemble around new viral RNA genomes e) Virus leaves the cells cloaked in plasma membrane specially coated with viral glycoprotein spikes – does not necessarily lyse the cell 2. The entire process occurs in the cytoplasm (other viruses might reproduce in the nucleus). Different viruses have evolved different mechanisms. C. Enveloped dsDNA virus - Herpes viruses (chickenpox, shingles, mononucleosis, cold sores, and genital herpes) reproduce in the cells nucleus 1. Can insert their DNA into the cells DNA as a provirus similar to a prophage in the lysogenic cycle 2. Different strains of the Herpes virus causes different disease D. Viruses that attack quickly repaired tissue are usually recoverable through mitosis while non-repairable tissues (eg. Nerves / polio virus) are much more severe E. How can we stop viruses? 1. Vaccines are important in these more sever cases because they prevent infection 2. Not to be confused with antibiotics, which are molecules that attack bacteria 3. Anitviral development is SLOW because of how hard it is to find a molecule that will kill the virus without killing the host cells F. Three major human viruses causing disease: influenza, cold viruses, herpes viruses XVI. Plant viruses are serious agricultural pests A. Most are RNA viruses B. Plants whose epidermal layer has been damaged by wind, injury, chill, or insects are more susceptible C. Insects, Farmers, Gardeners may spread these viruses (pruning shears and other tools) D. Infected plants may pass virus to offspring E. There are no cures for most viral plant diseases. Research has focused on prevention and selective breeding of resistant varieties. XVII. Emerging viruses threaten human health A. Predicted by the theory of evolution – “we live in evolutionary competition with microbes. There is no guarantee that we will be the survivors” – Joshua Lederberg, geneticist B. Some viruses 1. HIV – identified in the 1980’s 2. FLU – every year 3. EBOLA – identified in 1978 4. HANTAVIRUS C. New viruses can arise: 1. Through mutation of existing viruses – RNA viruses have VERY high mutation rates – no proofreading during replication! – Influenza 2. Spread of existing viruses to a new host species a) ~75% of human diseases originated in other animals (1) Hanatavirus common in rodents D. Viruses can spread like fire 1. HIV – unnoticed for decades before until blood transfusion technology, affordable travel, sexual promiscuity, and abuse of IV drugs enabled the virus to spread. XVIII. The AIDS virus makes DNA on an RNA template A. HIV is the virus that causes the disease AIDS 1. HIV – Human Immunodeficiency Virus 2. AIDS – Acquired Immune Deficiency Syndrome 3. Both terms describe the effect on the body – kills several kind of white blood cells (WBC’s) B. HIV is a retrovirus 1. Retroviruses synthesize DNA from an RNA template (retro = reverse) 2. Requires a special enzyme called reverse transcriptase (transcription in the reverse direction) C. The newly synthesized viral DNA can insert into the hosts DNA making it a provirus D. Review structure and behavior of HIV XIX. Virus research and molecular genetics are intertwined A. Scientists have a love-hate relationship with viruses 1. Some loves a) Hershey Chase Experiments on phage T2 concluding that DNA is the hereditary material leading to Watson-Crick model of DNA b) Viral reproduction has since helped show how genetic information flows from DNA to protein c) Gene delivery (gene therapy) 2. The hates a) Disease