3_7_08_Transcription_and_translation - Clicker Question...

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Unformatted text preview: Clicker Question Clicker According to eugenics, marriage is: According eugenics A) a union of two lines of property-descent. B) an experiment in breeding. C) the climax of human courtship. D) a way of fixing a certain status. E) all of the above. Where are we? Where Last time I talked about… – the discovery of DNA as the chemical that makes up the genome the – the eugenic movement in the United States which the defined people based on their genes. defined – Will we be prepared to accept and appreciate genetic Will diversity in the age of “designer babies”? diversity Today I will talk about… – how genes are replicated – how the information they contain are expressed through how the processes of transcription and translation. the – the chemistry of proteins. Why is the double helix structure so beautiful? beautiful? DNA Replication DNA DNA replication depends on the ability of bases to DNA undergo complementary base-pairing in a process known as DNA templating. DNA synthesis is “semiDNA conservative”, and each strand acts as a template for ”, a new strand. During replication, the strands separate and adenine During pairs with thymine, and cytosine pairs with guanine. pairs pairs The result is two identical daughter molecules— molecules— each one forms one chromosome and goes to one of each the daughter cells formed during mitosis. the Arthur Kornberg Discovered DNA Replication Is Mediated by Enzymes Not by a “Vital Force” “Vital “…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?" 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.” Arthur Kornberg Discovered DNA Replication Is Mediated by Enzymes Not by a “Vital Force” Mediated “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 To in vitro, Kornberg had in Kornberg to add to – An enzyme fraction An enzyme containing DNA polymerase – the four nucleotides the four (dATP, dGTP, dTPP and dCTP) and – A DNA template DNA Synthetic DNA and Synthetic Life Synthetic Using DNA polymerase it Using is now possible to synthesize DNA using any desired sequence as a template. template. This is the goal of Craig This Venter, founder of Synthetic Genomics, Inc. Synthetic You will synthesize copies You of your own DNA in lab this and next week. this http://www.syntheticgenomics.com/ http://www.etcgroup.org/en/ Synthetic Genomics Synthetic “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 genome of As a cell as the operating system and the cell cytoplasm of the cell as the hardware.” cytoplasm .” “Synthetic Genomics' goal is to modify the cell’s Synthetic operating system, design new genomes, to code for new types of cells with desired properties for the production of bioenergy or substitutes for petrochemicals.” petrochemicals.” Totipotency Totipotency Some cells Some (including embryonic stem cells) are capable of giving rise to all the cell types necessary to form a complete organism. These cells are called totipotent. totipotent Totipotency and Differential Transcription Totipotency • Probably all cells are totipotent Probably 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 In each cell type, only part of the In part code in the DNA is transcribed 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. cell Differential Transcription Differential For example, the gene for amylase is transcribed in cells of the For 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 of the glycolytic pathway are transcribed in all cells. enzymes The sry Gene is Not Expressed in All Cells at All Times at The sry gene is The sry 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 Anette Mayer finds RNA Anette transcripts of the sry transcripts 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: The Synthesis of RNA Transcription: The synthesis of RNA requires the enzyme RNA The RNA polymerase and ATP, UTP, CTP, and GTP, which and ATP UTP GTP which pair with T, A, G and C on the DNA, respectively. pair Transcription Transcription Eukaryotic RNA Eukaryotic polymerases do not bind directly to DNA, but must first bind to a protein protein transcription factor that factor recognizes a given promoter sequence on the on DNA. DNA Transcription Transcription 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 synthesis involves the splitting apart of the RNA two DNA strands and then the RNA polymerase RNA extends the newly formed RNA polymer. The RNA polymerase adds ribonucleotides at a The rate of about 30 nucleotides per second until it reaches a terminator sequence. terminator Once the RNA polymerase reaches the Once terminator sequence, the newly synthesized terminator the RNA separates from the DNA. RNA Alternative Splicing of RNA Leads to Isoforms of Proteins Isoforms Long sequences, known Long as introns, are removed introns are from the midst of the nascent RNA transcript. The remaining The sequences, known as exons, are spliced exons are together to form the messenger RNA. messenger As a result of alternative As splicing, a variety of splicing variety messenger RNAs can be formed from a single gene. DNA Is Transcribed into RNA DNA And the mRNA leaves the nucleus through the nuclear pores and enters the cytoplasm. Translation Translation Following the splicing Following of the RNA transcript, the mature messenger RNA messenger (mRNA) is recognized by the nuclear pore complex and transported to the cytoplasm where it is translated into protein translated by the ribosomes. ribosomes Gene Products Gene Most gene products are proteins, although Most some final products are RNAs. some Some of the gene products we have Some discussed include amylase, lipase, pepsinogen, trypsinogen, maltase, invertase, lactase, hemoglobin, membrane transporters on the small intestine and renal tubule, insulin, secretin, luteinizing hormone, follicle stimulating hormone, antidiuretic hormone, oxytocin, actin and myosin. hormone, 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 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 Affinity The study of affinity The began with Empedocles (≈ 450 Empedocles B.C.) who thought that chemicals had the qualities of love and love hate. To him, hate To chemical combination and decomposition was analogous to marriage and divorce, respectively. What Brings Two People Together? What Opposites Attract Birds of a Feather Flock Together Together Love and Chemistry Love Believe it or not, the Believe laws of attraction are the same with chemicals. chemicals. For some chemicals, For “opposites attract.” “opposites For other chemicals, For “Birds of a feather flock together.” flock Affinity Between Two Substances: Birds of a Feather Flock Together Birds Hippocrates concluded Hippocrates that only chemicals that shared a kinship with each other combined to each form compounds. This thinking has been captured in terms like hydrophilic (waterhydrophilic lloving) and oving) hydrophobic (water(waterhating)! Affinity Between Two Substances: Opposites Attract Opposites By contrast, By Heraclitus argued that chemicals with opposite properties attract and thus attract form compounds. Affinity Between Two Substances Affinity Hippocrates was correct Hippocrates for the interactions between two polar (hydrophilic) molecules (hydrophilic) or two nonpolar or (hydrophobic) (hydrophobic) molecules. molecules. Heraclitus was right Heraclitus when it came to the interactions between charged chemicals. charged Neither theory was allencompassing. Rules Necessary to Determine Affinities Between Molecules Affinities Affinity can be deduced by very simple chemical rules Affinity based on a firm foundation of quantum mechanics. quantum Rule I: In a given row of the periodic table, the last In element is called a noble gas. It is very stable and it noble It stable has little or no attraction for any other chemical. Rule II: In a given row in the periodic table, the more In protons an atom has, the greater its attraction for protons electrons and the more electronegative it will be. electronegative Rule III: Atoms will lose, gain or share electrons in Atoms lose gain share order to obtain the electron configuration of the nearest noble gas. nearest The Electronegativity of Atoms The The atoms on the left side of the periodic table The 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 positively charged. positively The atoms on the right side of the periodic table The 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 charged. negatively Polar and Nonpolar Molecules Polar 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 the sharing, C forms polar or nonpolar molecules. nonpolar C and H have equal affinity and equal for electrons so CH bonds are nonpolar. are O has a greater affinity for greater electrons than C or H, so OH and CO bonds are polar. and Methane (nonpolar) and Water (polar) Methane Oil (nonpolar) and Vinegar (polar) Oil Atoms that Do Not Share Electrons, But Rather Lose or Gain Them Become Charged Become For Charged Atoms or Molecules, Opposites Attract For Protein: A Polymer of Polar, Nonpolar and Charged Amino Acids Nonpolar Affinity Between Two Molecules In order to obtain the specific binding that In specific enzymes and peptide hormones show, they make use of 20 different amino acids. 20 Some amino acids are hydrophobic and Some have an affinity for hydrophobic substrates; other amino acids are substrates other hydrophilic and have an affinity for hydrophilic substrates; positively-charged hydrophilic positively-charged amino acids bind negatively-charged substrates and negatively-charged amino substrates acids bind positively-charged substrates. positively-charged Proteins Proteins The linear The polypeptide, with all its polar, nonpolar and charged amino acids, folds into a threethreedimensional dimensional structure so it can function. can Biosynthetic and Hydrolytic Enzymes Biosynthetic enzymes have the affinity to bind Biosynthetic affinity two substrates in two pockets formed by amino acids. The binding induces a mechanical amino The movement of the enzyme which causes the two movement 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 high affinities for product no the enzyme and they dissociate as the product so the enzyme can be recycled to perform another recycled synthesis. Hydrolytic enzymes work in the reverse direction Hydrolytic and one of the substrates is water. and Heat: Activation Energy Without the enzyme, two Without substrates would have to collide into each other by chance. chance. The chance of collision is The increased by heating up the substrates so they move faster. The thermal energy added is known as the activation energy (EA). activation By binding the substrates and By bringing them close enough to bond, there is no need to heat up the substrates. This is why enzymes can This catalyze reactions at ambient temperatures. Proteins Are Synthesized on Ribosomes Ribosomes A Transfer RNA Binds an Amino Acid Transfer Robert Holley (USDA/Cornell) discovered tRNA. Aminoacyl-tRNA Synthetase Aminoacyl-tRNA Each tRNA, with a Each specific anticodon, anticodon binds to a specific amino acid with the help of a specific aminoacylof tRNA synthetase. tRNA This step requires ATP. This is the first step in This translating the genetic code encoded in the nucleic acids to a protein sequence. Rosetta Stone: The Code is Cracked Rosetta Translation of the 3 Translation nucleotide codons into amino acids (three letter abbreviations). Transfer RNA Transfer One end of a tRNA binds a specific amino acid. The One other end of the tRNA contains a sequence of three nucleotides (UAC), known as an anticodon, which is anticodon which complementary to and thus can bind with the three complementary nucleotide codon (AUG) of a mRNA in the ribosome. nucleotide Protein Synthesis: Ribosome The small subunit of the ribosome contains one The binding site for mRNA and two for tRNA. One tRNA binding site is called the peptidyl‑tRNA One binding site (P-site). This site holds the tRNA that is binding ). tRNA linked to the growing end of the polypeptide chain. growing The other tRNA binding site is called the The aminoacyl‑tRNA binding site (A-site) and it holds the binding and incoming tRNA bound to an amino acid. incoming Protein Synthesis: Initiation Protein synthesis is initiated when Protein methionine‑tRNA binds to the P‑site. Pari passu, Pari passu the small subunit binds a mRNA. The small subunit of the ribosome, containing the The methionine‑tRNA then moves down the mRNA in search of the AUG codon (start codon). search ). Then the small and large subunits bind together, Then completing the formation of the ribosome. Protein synthesis continues as the next Protein aminoacyl‑tRNA binds to the A‑site. aminoacyl‑tRNA Chain Elongation and Proofreading In order to add an amino acid to the polypeptide chain, In GTP must be hydrolyzed. GTP This will only happen if the GTP is in the A-site for a This sufficient amount of time. The GTP will only remain in the A-site for a long The enough time if there is perfect matching between the codon and anticodon. the If the match is not perfect, the aminoacyl-tRNA If complex is released from the ribosome before the GTP is hydrolyzed. The requirement for a correct binding before the GTP The is hydrolyzed results in a proofreading mechanism proofreading that ensures the correct sequence of amino acids in a protein. protein. Protein Synthesis: Energy Intensive Protein After GTP hydrolysis, the polypeptide chain is After uncoupled from the tRNA molecule in the P-site and linked to the amino group of the aminoacyl‑tRNA in the A-site to form a peptide bond. The free tRNA in the P-site is released from the The ribosome as the peptidyl‑tRNA in the A-site is translocated to the P-site. This movement also requires the hydrolysis of GTP. requires In this way, the ribosome moves down the mRNA In three nucleotides at a time as it synthesizes a protein. The hydrolysis of three nucleoside triphosphates is The necessary for the addition of each amino acid to a protein and consequently, protein synthesis is an energy intensive process. energy Termination of Protein Synthesis Termination Protein synthesis is terminated Protein when the ribosome reaches a stop codon (UAA, UAG or stop UGA). The stop codon causes a The ribozyme, which is an enzyme which in the ribosome made of RNA, to add a water molecule RNA, instead of an amino acid to the nascent polypeptide. The polypeptide chain is freed The from the tRNA and released from the ribosome. Protein Synthesis Protein Approximately 10 Approximately amino acids are incorporated into a protein per second and thus an average protein, which has 200-600 amino acids, is synthesized in a ribosome in about 20‑60 seconds. 20‑60 Protein Structure Protein The resulting protein has The protein the correct amino acid sequence so it can fold sequence into a three dimensional structure with pockets structure with the correct polarity and charge to bind and substrates, receptors, substrates receptors hormones, etc with hormones etc appropriate affinity. affinity Protein Targeting: Molecular Zip Codes Codes Once a protein is synthesized it must be targeted to its Once correct location in the cell. This intracellular sorting is accomplished by the presence This of a signal peptide, a llinear stretch of 15‑60 amino acids inear signal that is often but not always removed from the mature protein once translocation into the targeted organelle is completed. The importance of the individual signal peptides for protein The targeting has been demonstrated by putting the signal sequence on another peptide using genetic engineering techniques. The targeting of a protein can then be controlled artificially. Such experiments clearly show that the peptide, which forms a molecular zip code is molecular necessary and sufficient for correct targeting. Post-translational Modifications Post-translational There are many postThere posttranslational modifications (adding phosphate, lipid, sugar, etc.) that can be made to proteins that change their properties. change In collagen, the extracellular In collagen the protein of connective tissue, the prolines are converted to hydroxyprolines, posthydroxyprolines, translationally. This step requires translationally. vitamin C. vitamin Without vitamin C, collagen of Without connective tissue is defective and gums bleed and bones fall apart, a condition known as scurvy. scurvy Protein Turnover Protein The introduction of stable and rare isotopes of H, O, The N, and S by Rudolf Schoenheimer (1942) made possible the astonishing results that “all possible all constituents of living matter, whether functional or structural, of simple or of complex constitution, are in a steady state of flux.” constitution, .” Prior to his work, it seemed reasonable that living Prior 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. most Living matter is constantly being built up and broken Living down, and each molecule has a lifetime that is less than that of the organism itself. than Proteolysis: Recycling of Amino Acids Acids All proteins are eventually degraded in the All cell by proteases. In this controlled intracellular digestion, In controlled the various proteases hydrolyze the substrate proteins into free amino acids. As free amino acids they are attached to As their tRNA and participate again in protein synthesis. Amino acids that are not recycled produce ammonia, which is converted to urea in the ammonia which urea liver and eliminated from the blood by the kidney. kidney. Inhibition of the Proteolysis of Skeletal Muscle Proteins Muscle 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 Nucleus to the Time of Its Death of Hereditary Diseases Result From the Inability to Produce a Functional Enzyme Enzyme Archibald Garrod (1909) Archibald proposed that hereditary diseases, iincluding diseases ncluding phenylketonuria (PKU) and albinism were due to inborn errors in metabolism that resulted metabolism from a person’s inability to synthesize a particular functional enzyme. enzyme Hereditary Diseases: Inborn Errors in Metabolism Errors Sickle-cell Anemia Results From a Mutant Form of Hemoglobin Mutant The Relationship between a Gene and a Protein Protein A gene codes for a polypeptide gene chain. A protein is composed of one or protein more polypeptide chains. The polypeptide chain may act The as a hormone (insulin), hormone receptor (insulin receptor), enzyme (amylase), transport protein (glucose transporter), protein movement protein (actin and movement myosin) or a structural protein structural (collagen). (collagen). Proteins Are Microscopic Machines that Perform Almost All the Functions We Associate with Being Alive Being Additional Resources Additional Nova: Cracking the Code of Life Nova: ...
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This note was uploaded on 04/04/2009 for the course BIO G 110 taught by Professor Wayne,r. during the Spring '07 term at Cornell.

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