3-15-10 Transcription and translation-color

3-15-10 Transcription and translation-color - A Drama PCR...

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Unformatted text preview: A Drama PCR Song Central Dogma Obsession PCR Song GCTA Clicker Question According to eugenics, eugenics marriage is defined as _____________. A) a union of two lines of un li propertyproperty-descent B) an experiment in breeding C) the climax of human courtship and love D) a way of fixing a certain status E) a necessity for farming BioRad GTCA PCR Song Where are we? Last Last time I talked about… – the discovery of DNA as the chemical that is passed from parents to offspring and the chemical that contains an individual’s unique genetic information. – the eugenics movement in the United States which defined people based on their genes, and defined marriage as an experiment in breeding marriage as an experiment in breeding. – Will we be prepared to accept and appreciate genetic diversity in the age of Newgenics and “designer babies” which may redefine parents as product developers? The Science of Marriage Today Today I will talk about… – how DNA is replicated. – how the information DNA contains is expressed through the processes of transcription and translation. – the chemistry of proteins. www.timesonline.co.uk 1 …the Greatest of them all is Love …and in today’s lecture I will discuss the similarities between chemistry and love. Jim Watson and a Collaborator Who Got a DNA Tattoo When He Received His Ph.D. Another DNA Tattoo Another DNA Tattoo Teaching Biology through Tattoos? 2 DNA’s Double Helix is Beautiful Why is the double helix structure so beautiful? DNA Replication Arthur Kornberg Discovered DNA Replication Is Mediated by Enzymes Not by a “Vital Force” “…we have in the Watson and Crick proposal a mechanical model of replication, we may at this point pose the question: "What is the chemical mechanism by which this super molecule is built up in the cell? molecule is built up in the cell?" Some sixty years ago the alcoholic fermentation of sugar by a yeast cell was a “vital” process inseparable from the living cell, but through the Buchner discovery of fermentation in extracts and the march of enzymology… we understand fermentation by yeast as a…sequence of integrated chemical reactions.” DNA replication depends on the ability of bases to DNA replication depends on the ability of bases to undergo undergo complementary base-pairing in a process baseknown as DNA templating. DNA synthesis is “semiDNA semiconservative”—each strand acting as a template for a new strand. During During replication, the strands separate and adenine adenine pairs pairs with thymine, and cytosine pairs with guanine. pairs The result is two identical daughter molecules— two each one forms one chromosome and each one goes to one of the daughter cells formed during mitosis. 3 Arthur Kornberg Discovered DNA Replication Is Mediated by Enzymes Not by a “Vital Force” “Five years ago the synthesis of DNA was also regarded as a "vital" process. Some people considered it useful for biochemists to examine the combustion chambers of the cell [mitochondria], but tampering with the very genetic apparatus itself would surely produce nothing but disorder. These gloomy predictions were not justified then, nor are similar pessimistic attitudes justified now….” “The enzyme we are studying is thus unique in present experience in taking directions from a template.” To get DNA synthesis in vitro, Kornberg had to add – An enzyme fraction enzyme fraction containing DNA polymerase – the four nucleotides (dATP, dGTP, dTPP and dCTP) – A DNA template that the enzyme can “read” Helping a Distressed Individual In his book entitled, For the Love of Enzymes, Arthur hi Kornberg wrote, “there is the story…of a surgeon who, while jogging around a lake, spotted a man drowning. He pulled off his clothes, dove in, dragged the victim ashore, and resuscitated him. He resumed his jogging, only to see another man drowning. After he dragged the second one out and got him breathing again, he wearily resumed his jogging.” jogging.” Having Having a Prepared Mind to Reduce Distress in the First Place “Soon he saw several more drowning. He also saw a professor of biochemistry nearby, absorbed in thought. He called to the biochemist to go after one while he went after another. When the biochemist was slow to respond, he asked him why he wasn’t doing something. The biochemist said: ‘I am doing something. I’m desperately trying to figure out who’s throwing all these people in the lake.’” 4 Synthetic DNA and Synthetic Life Using Using DNA polymerase it is now possible to synthesize DNA using any desired sequence as a template template. This This is the goal of Craig Venter, founder of Synthetic Genomics, Inc. You You will synthesize copies (i.e. make clones) of your own DNA in lab. http://www.syntheticgenomics.com/ http://www.etcgroup.org/en/ Synthetic Genomics “Synthetic genomics is a new field of science that involves the design and assembly of genes and gene pathways and whole chromosomes from chemical components of DNA.” “As a computer analogy, we view the As computer analogy we view the genome genome of a cell as the operating system and the cytoplasm of the the cell as the hardware.” “Synthetic “Synthetic Genomics' goal is to modify the cell’s operating system, design new genomes, to code for new types of cells with desired properties for the production of bioenergy or substitutes for petrochemicals.” ETC Totipotency Some cells (including embryonic stem cells) are capable of giving capable of giving rise rise to all the cell types necessary to form a complete organism. These cells are called totipotent totipotent. In the 1950s, F. C. Steward at Cornell isolated single In cells from carrots. When provided with the right hormones, the cells formed embryos, which grew into carrot plants. He concluded that each and every cell is totipotent He totipotent and has the potential to give rise to a whole organism. 5 Totipotency and Differential Transcription • Probably all cells are totipotent totipotent and contain a full complement of genes. Cell differentiation and Cell the resulting phenotype results phenotype from differential transcription or differential differential gene expression differential gene expression. In In each cell type, only part of the part code in the DNA is transcribed transcribed into into RNA molecules. These RNAs are then translated into the amino translated acid sequences that will give the specific proteins required for each cell type and our own individuality. Differential Transcription For example, the gene for amylase is transcribed in cells of the amylase is salivary gland, the gene for pepsinogen is transcribed in the pepsinogen chief cells, the genes for trypsinogen is transcribed in the trypsinogen exocrine cells of the pancreas; the genes for actin and myosin actin myosin are transcribed in the muscle cells, and the genes for the enzymes enzymes of the glycolytic pathway are transcribed in all cells. The sry Gene is Not Expressed in All Cells at All Times The sry The sry gene is transcribed in the gonads for a couple of days in the 6th-7th week of embryonic development and leads to the formation of testes the formation of testes. Anette Anette Mayer finds RNA RNA transcripts of the sry sry gene in the frontal cortex, the temporal cortex and the hypothalamus of male cadavers, indicating that “maleness” may also be genetically expressed in the cells of the brain. brain Transcription: Transcription: The Synthesis of RNA Transcription Eukaryotic RNA polymerases do not bind directly to DNA, but must first bind to a protein protein transcription factor (TF) that recognizes a given promoter promoter sequence on the on DNA. TF The synthesis of RNA requires the enzyme RNA RNA polymerase and ATP, UTP, CTP, and GTP, which and UTP pair with T, A, G and C on the DNA, respectively. 6 Androgen Receptors transcription factors The androgen receptors The are ligand-activated ligandthat regulate gene that expression. That is, when they bind That androgen, they move into the nucleus and turn on genes. Transcription Promoters Promoters are sequences of DNA, upstream from the transcribed region of the gene, which cause either prodigious or small quantities of RNA to be transcribed. RNA RNA synthesis involves the splitting apart of the two DNA strands and then the RNA th th RNA polymerase polymerase extends the newly formed RNA polymer. The The RNA polymerase adds ribonucleotides at a rate of about 30 nucleotides per second until it reaches a terminator sequence. terminator Once Once the RNA polymerase reaches the terminator sequence, the terminator newly synthesized RNA separates from the DNA. TF Alternative Splicing of RNA Leads to Isoforms of Proteins Long Long sequences, known as introns, are removed introns from the midst of the nascent RNA transcript. The remaining The remaining sequences, sequences, known as exons exons, are spliced together to form the messenger RNA. As As a result of alternative alternative splicing, a variety of messenger RNAs can be formed from a single gene. DNA DNA Is Transcribed into RNA And the mRNA leaves the nucleus through the nuclear pores and enters the cytoplasm. Translation Following the splicing of the RNA transcript, the mature mature messenger RNA (mRNA) is recognized by the nuclear pore complex and transported to the cytoplasm where it is translated translated into protein protein by by the ribosomes. ribosomes Gene Gene Products Most Most gene products are proteins, although some final products are some RNAs, RNAs, including ribosomal RNA, transfer RNA and regulatory RNAs known as micro RNAs. RNAs. Some Some of the gene products we have discussed include amylase, lipase, pepsinogen, trypsinogen, pepsinogen, trypsinogen, maltase, invertase, invertase, lactase, hemoglobin, myoglobin, myoglobin, membrane transporters on the small intestine and renal tubule, insulin, secretin, luteinizing secretin, hormone, follicle stimulating hormone, antidiuretic hormone, oxytocin, actin, oxytocin, actin, myosin and rubisco. ubisco. 7 Enzymes and Peptide Hormones • Enzymes can be extremely specific catalysts for a given substrate. To be so specific, they must have a high affinity for the substrate. In 1894 Emil Fi Fischer wrote about enzymes and substrates, “The one may be said to fit into the other as a key fits into a lock.” • Likewise peptide hormones must have specific chemical properties to bind with high affinity to receptors. Affinity Between Two Substances The study of affinity began with Empedocles (≈ 450 B.C.) who thought that chemicals had the chemicals had the qualities love qualities of love and hate hate. To him, chemical combination and decomposition was analogous to marriage and divorce, respectively. What What Brings Two People Together? Opposites Attract Birds Birds of a Feather Flock Together Love and Chemistry Believe Believe it or not, the laws of attraction are the same with chemicals. For For some chemicals, “opposites attract.” For For other chemicals, “Birds of a feather flock together.” 8 Affinity Between Two Substances: Birds of a Feather Flock Together Hippocrates concluded that only chemicals that chemicals shared a kinship with each other combined to each other combined to form form compounds. This thinking has been captured in terms like hydrophilic (waterhydrophilic (waterloving) and hydrophobic (waterhydrophobic (waterhating)! Affinity Affinity Between Two Substances: Opposites Attract By contrast, Heraclitus argued that chemicals chemicals with opposite properties attract and thus form compounds. Affinity Affinity Between Two Substances Hippocrates Hippocrates was correct for the interactions between two polar polar (hydrophilic) molecules or two nonpolar (hydrophobic) (hydrophobic) molecules. Heraclitus Heraclitus was right when it came to the interactions between charged charged chemicals. Neither Neither theory was allallencompassing. Rules Necessary to Determine Affinities Between Molecules Affinity can Affinity can be deduced by very simple chemical rules based on a firm foundation of quantum mechanics. quantum Rule Rule I: In a given row of the periodic table, the last element is called noble gas It is very stable and it element is called a noble gas. It is very stable and it has has little or no attraction for any other chemical. Rule Rule II: In a given row in the periodic table, the more more protons an atom has, the greater its attraction for greater electrons and the more electronegative it will be. and electronegative Rule Rule III: Atoms will lose, gain or share electrons in lose gain share order to obtain the electron configuration of the nearest nearest noble gas. 9 The Electronegativity of Atoms The The atoms on the left side of the periodic table try to lose electrons (become oxidized) so they will have an electronic configuration like the previous noble gas. When they lose an electron they become electron they become positively positively charged. The The atoms on the right side of the periodic table try to gain electrons (become reduced) until they have the electronic configuration of the next noble gas. When they gain an electron, they become negatively negatively charged. Polar and Nonpolar Molecules Carbon Carbon is in the middle of the row and shares shares electrons with other atoms in order to obtain the electronic configuration of neon. Depending of the equality of Depending of the equality of the the sharing, C forms polar polar or nonpolar or nonpolar molecules. C and H have equal affinity and equal for electrons so CH bonds are nonpolar. O has a greater affinity for has greater electrons than C or H, so OH and CO bonds are polar. Methane (nonpolar) and Water (polar) Oil (nonpolar) and Vinegar (polar) cis Carbon Tends to Share Electrons Oxygen Tends to Hoard Electrons 10 Atoms that Do Not Share Electrons, But Rather Lose or Gain Them Become Charged Protein: A Polymer of Polar, Nonpolar and Charged Amino Acids For Charged Atoms or Molecules, Opposites Attract Affinity Between Two Molecules In In order to obtain the specific binding specific that enzymes and peptide hormones show, they make use of 20 different 20 amino acids. Some amino acids are hydrophobic and have have an affinity for hydrophobic hydrophobic substrates; other amino acids are hydrophilic and have an affinity for hydrophilic hydrophilic substrates; positivelypositivelycharged amino acids bind negativelynegativelycharged substrates and negativelynegativelycharged amino acids bind positivelypositivelycharged substrates. Positively charged Negatively charged Proteins The linear polypeptide, with all its polar, nonpolar and charged amino acids, folds into a threethreedimensional structure so it can function. Roderick MacKinnon won the Nobel Prize for Elucidating the Structure of a Potassium Potassium Channel using the Synchrotron at Cornell 11 Biosynthetic and Hydrolytic Enzymes Biosynthetic Biosynthetic enzymes have the affinity to bind two affinity bind substrates in two pockets formed by amino acids. Th The binding induces a mechanical movement of the enzyme which causes the two substrates to get so close together that they begin to share electrons with each other and make a bond. The bonded substrates, which is now known as the product, no longer have product high affinities for the enzyme and they dissociate as the product so the enzyme can be recycled to recycled perform another synthesis. Hydrolytic Hydrolytic enzymes like invertase work in the reverse direction and one of the substrates is water. Heat: Activation Energy Without Without the enzyme, two substrates would have to collide into each other by chance. The The chance of collision is increased by heating up the substrates so they move faster. The thermal energy added is known as the activation activation energy (EA). By By binding the substrates and bringing them close enough to bond, there is no need to heat up the substrates. This This is why enzymes can catalyze reactions at ambient temperatures— temperatures—a characteristic of life. 12 Proteins Are Synthesized on Ribosomes A Transfer RNA Binds an Amino Acid Robert Holley Robert Holley (USDA/Cornell) discovered tRNA. AminoacylAminoacyl-tRNA Synthetase Each Each tRNA, with a specific anticodon, anticodon binds to a specific amino acid with the help of specific aminoacyl of a specific aminoacyltRNA synthetase. This This step requires ATP. This This is the first step in translating the genetic code encoded in the nucleic acids to a protein sequence. Rosetta Rosetta Stone: The Code is Cracked Translation of the 3 nucleotide codons codons into into amino acids (three letter abbreviations). 13 Transfer RNA Genetic Genetic Code: The Relationship Between the Codon and the Amino Acid One end of a tRNA binds a specific amino acid. The other end of the tRNA contains a sequence of three nucleotides (UAC), known as an anticodon, which is anticodon complementary complementary to and thus can bind with the three three nucleotide codon (AUG) of a mRNA in the ribosome. Protein Synthesis: Ribosome Protein Protein Synthesis: Initiation The The small subunit of the ribosome contains one binding site for mRNA and two for tRNA. One One tRNA binding site is called the peptidyl-tRNA peptidylbinding site (P-site). This site holds the tRNA that is tRNA linked to the growing end of the polypeptide chain. growing The The other tRNA binding site is called the aminoacylaminoacyl-tRNA binding site (A-site) and it holds the binding incoming incoming tRNA bound to an amino acid. Protein Protein synthesis is initiated when methionine-tRNA binds to methioninethe P-site. Pari passu, the small subunit binds a mRNA. PPari The The small subunit of the ribosome, containing the methioninemethionine-tRNA then moves down the mRNA in search of the AUG codon (start codon). Then Then the small and large subunits bind together, completing the formation of the ribosome. Protein Protein synthesis continues as the next aminoacyl-tRNA binds aminoacylto the A-site. A- Chain Elongation and Proofreading In In order to add an amino acid to the polypeptide chain, GTP must be hydrolyzed. This This will only happen if the GTP is in the A-site for a Asufficient amount of time. The The GTP will only remain in the A-site for a long Aenough time if there is perfect matching between enough time if there is perfect matching between the the codon and anticodon. If If the match is not perfect, the aminoacyl-tRNA aminoacylcomplex is released from the ribosome before the GTP is hydrolyzed. The The requirement for a correct binding before the GTP is hydrolyzed results in a proofreading mechanism proofreading that ensures the correct sequence of amino acids in a protein. 14 Protein Synthesis: Energy Intensive GTP Hydrolysis After After GTP hydrolysis, the polypeptide chain is uncoupled from the tRNA molecule in the P-site and Plinked to the amino group of the aminoacyl-tRNA in aminoacylthe A-site to form a peptide bond. AThe The free tRNA in the P-site is released from the Pribosome as the peptidyl-tRNA in the A-site is ltranslocated to the P-site. This movement also Prequires the hydrolysis of GTP. In In this way, the ribosome moves down the mRNA three nucleotides at a time as it synthesizes a protein. The The hydrolysis of three nucleoside triphosphates is necessary for the addition of each amino acid to a protein and consequently, protein synthesis is an energy intensive process. Termination of Protein Synthesis GTP Hydrolysis GTP Hydrolysis Protein Protein synthesis is terminated when the ribosome reaches a stop stop codon (UAA, UAG or UGA). The stop codon causes The stop codon causes a ribozyme, which ribozyme, which is an enzyme enzyme in the ribosome made of RNA, to add a water molecule instead of an amino acid to the nascent polypeptide. The The polypeptide chain is freed from the tRNA and released from the ribosome. Protein Protein Synthesis Approximately 10 amino acids are incorporated into a protein per second protein per second and and thus an average protein, which has 200-600 200amino acids, is synthesized in a ribosome in about 2020-60 seconds. Protein Structure The resulting protein has protein the correct amino acid amino sequence so it can fold into a three dimensional three structure structure with pockets pockets with the correct polarity and charge to bind bind substrates, receptors, receptors hormones hormones, etc with appropriate affinity. affinity 15 Protein Targeting: Molecular Zip Codes Once Once a protein is synthesized it must be targeted to its correct location in the cell. This This intracellular sorting is accomplished by the presence li th of a signal peptide or transit signal transit peptide, which is a linear stretch of 15-60 amino acids 15that is often but not always removed from the mature protein once translocation into the targeted organelle is completed. Protein Protein Targeting: Molecular Zip Codes The The importance of the individual signal peptides for protein targeting has been demonstrated by putting the signal sequence on another peptide using genetic another peptide using genetic engineering engineering techniques. The The targeting of a protein can then be controlled artificially. Such Such experiments clearly show that the peptide, which forms a molecular molecular zip code is necessary and sufficient for correct targeting. PostPost-translational Modifications There There are many postposttranslational modifications (adding phosphate, lipid, sugar, etc.) that can be made to proteins that change their properties. change In collagen the extracellular In collagen, the extracellular protein protein of connective tissue, the prolines are converted to hydroxyprolines, postposttranslationally. This step requires vitamin vitamin C. Without Without vitamin C, collagen of connective tissue is defective and gums bleed and bones fall apart, a condition known as scurvy. scurvy Protein Turnover The The introduction of stable and rare isotopes of H, O, N, and S by Rudolf Schoenheimer (1942) made possible the astonishing results that “all constituents of living matter, whether functional or structural, of simple or of complex constitution, are in a steady state of flux.” Prior Prior to his work, it seemed reasonable that living matter was like a machine that used nutritive substances as fuel to run the machine and the excretions were the part of the food that were not used. After all, most people’s size stays constant for most of their lives. Living Living matter is constantly being built up and broken down, and each molecule has a lifetime that is less than that of the organism itself. www.limestrong.com 16 Materially, We are not always the Same Person we Were when this Class Began Today, You Are What You Ate Yesterday Proteolysis: Recycling of Amino Acids All All proteins are eventually degraded in the cell by proteases. In In this controlled intracellular digestion, controlled the various proteases hydrolyze the substrate proteins into free amino acids. As As free amino acids they are attached to their tRNA and participate again in protein synthesis. Amino Amino acids that are not recycled produce ammonia ammonia, which is converted to urea in the urea liver and eliminated from the blood by the kidney. Inhibition of the Proteolysis of Skeletal Muscle Proteins Muscle mass depends on the balance of the synthesis of actin and myosin and their breakdown. β-hydroxy-βmethylbutyrate (HMB) inhibits the proteolysis (catabolism or breakdown) of these muscle proteins. HMB is a natural breakdown product of the amino acid, leucine. The Life Cycle of a Protein: From the Time It Was a Twinkle in the in the Nucleus Nucleus to the Time of Its Death Hereditary Diseases Result From the Inability to Produce a Functional Enzyme Archibald Garrod (1909) proposed that hereditary hereditary diseases, including phenylketonuria phenylketonuria (PKU) and albinism were due to inborn inborn errors in metabolism that resulted from a person’s inability to synthesize a particular functional enzyme. enzyme 17 Hereditary Diseases: Phenylketonuria, Alkaptonuria, Albinism—Inborn Errors in Albinism— Metabolism Hereditary Disorders or Conditions and Intersexuality: Dihydrotestosterone (DHT) Deficiency Hereditary Disorders or Conditions and Intersexuality: Adrenal Hyperplasia SickleSickle-cell Anemia Results From a Mutant Form of Hemoglobin The Relationship between a Gene and a Protein A gene codes for a polypeptide gene chain. A protein is composed of one or protein more polypeptide chains. The The polypeptide chain may act as a hormone (insulin), hormone receptor (insulin receptor (insulin receptor), enzyme (amylase), transport enzyme (amylase), transport protein (glucose transporter), movement movement protein (actin and myosin) or a structural protein structural (collagen). Proteins Are Microscopic Machines that Perform Perform Almost All the Functions We Associate with Being Alive 18 Gene Therapy: Replace a Defective Gene with another Gene that Produces a Functional Enzyme Rational Drug Design: Use X-Ray Diffraction or XComputer Modeling to Predict the Structure of a Protein in Order to Design Drugs that with Interact with it Identify and Improve Activity of Proteins Involved in Hydrogen Production for Biofuels 19 Additional Resources Nova: Cracking the Code of Life 20 ...
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This document was uploaded on 11/09/2010.

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