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Central Dogma of Biology DNAmRNAprotein DNA TRANSCRIBES to mRNA Process is called transcription mRNA TRANSLATES to proteins Process is called translation mRNA actually makes amino acids, which come together to make proteins Nucleic Acids RNA Single Strand Ribose sugar A=U G=C Uracil is the nitrogenous base used instead of THYMINE DNA Double strand Deoxyribose sugar A=T G=C DNA parent strand makes 2 daughter strands…one fast, smart daughter strand (leading) and one, slower, nofast daughter strand (lagging) Leading strand (runs 3’ to 5’) Lagging strand (runs 5’ to 3’) In the 1950’s, no one knew how DNA was replicated… 3 possibilities Conservative replication One completely new helix made from old one Semi-conservative replication New molecule of DNA would contain one old strand and one new strand Dispersive replication Each new molecule of DNA would be made out of old bits and new bits 1958 Meselsohn and Stahl Mathew Meselsohn and Franklin Stahl Used E. coli bacteria (common, harmless bacteria in human gut) Grew E. coli in food source that contained ammonium chloride for a source of nitrogen Experiment relied on variation in structure of nitrogen atoms (used isotope of nitrogen…N-15) 7 protons but neutrons vary=isotope=lil bits a radiation given off so they are traceable (ISOTOPES) Most have 7 neutrons so relative atomic mass is 14 Some have 8 neutrons so relative atomic mass is 15 Bacteria used the N-15 to make their DNA They were able to divide many times to make many copies of their DNA with the N-15 isotope Nearly all their DNA only contained this heavy N-15 isotope This DNA is heavier than normal DNA with normal N-14 Some of these new bacteria were transferred to another petri dish containing a food source with lighter N-14 Some were left to replicate just once (50 minutes) and others left long enough for just two, three and four times of replication DNA was extracted from this group Samples were placed in Cesium chloride and spun in a centrifuge The heavier the DNA, the closer to the bottom of the tube it came to rest Results How does DNA replicate itself? Template mechanism DNA Replication Process of copying the DNA molecule What phase of the CELL CYCLE? S-phase…. 2 strands of double helix separate (Unzips) Each strand acts as a negative for making the new complementary strand Nucleotides line up one by one following base pairing rules Enzymes (DNA Polymerase and DNA Ligase) link nucleotides together to form 2 new DNA strands called the daughter strands DNA Replication Simplified Unzip parent DNA Hydrogen bonds b/t bases break Nucleus contains ACTIVATED nucleotides (a little bit different than normal nucleotides) Contain 2 EXTRA phosphate groups (total of 3 now) Add activated nucleotides to the 2 template strands of DNA Enzyme connects the innermost phosphate of the activated nucleotide to the nucleotide already on the growing strand Two extra phosphate are released back into the nucleus DNA parent strand makes 2 daughter strands…one fast, smart daughter strand (leading) and one, slower, not-so-fast daughter strand (lagging) Leading strand (runs 3’ to 5’) Lagging strand (runs 5’ to 3’) Attach fragments on lagging strand Origins of Replication Specific site on DNA where replication begins DNA Helicase: enzyme that binds to origin site and unwinds DNA in both directions Copying goes outward in both directions making replication “bubbles” Parent strands open up as daughter strands grow on both sides Eukaryotic DNA has many origins of replication on a single DNA strand Makes copying faster Eventually bubbles merge making two new strands Each new strand has a part from the original and a part from the new Semi-conservative replication Means that parent of the original DNA double helix is always kept as part of the new DNA copies DNA Polymerases Enzymes Make covalent bonds between nucleotides of the new strands Fast, accurate process Error only one in a billion nucleotides Brings over ACTIVATED nucleotides to unzipped DNA strand and drops them off More Players in DNA Replication DNA polymerase can only read a strand that is running 3-prime to 5prime… New strand is built running from 5’ to 3’ DNA polymerase works non-stop adding nucleotides onto the strand that runs in the 3’ to 5’ direction Therefore, Only one strand is made by a smooth, and continuous process… The other strand is put together in bits and pieces… Each little section of nucleotides is called an “Okazaki Fragment” These are then “glued” together to make one, continuous strand in the end by another enzyme… DNA Ligase Important Enzymes DNA Helicase unzips DNA Polymerase Adds nucleotides DNA Ligase Attaches/glues Central Dogma of Biology DNAmRNAprotein DNA TRANSCRIBES to mRNA Process is called transcription mRNA TRANSLATES to proteins Process is called translation mRNA actually makes amino acids, which come together to make proteins DNAmRNAamino acids/polypeptide chain (Proteins) DNA codes for an RNA strand The every 3 bases on the RNA strand code for a specific amino acid CODON: three sequential bases that code for a specific a.a. (20 a.a. total) Amino acid are strung together to make a protein (primary structure) Change DNA will change RNA which will change amino acids, which change protein Legend: Transcription of DNA to RNA to protein: This dogma forms the backbone of molecular biology and is represented by four major stages. 1. The DNA replicates its information in a process that involves many enzymes: replication. 2. The DNA codes for the production of messenger RNA (mRNA) during transcription. 3. In eukaryotic cells, the mRNA is processed (essentially by splicing) and migrates from the nucleus to the cytoplasm. 4. Messenger RNA carries coded information to ribosomes. The ribosomes "read" this information and use it for protein synthesis. This process is called translation. Ala: Alanine Phe: Phenylalanine Lys: Lysine Pro: Proline Thr: Threonine Cys: Cysteine Gly: Glycine Leu: Leucine Gln: Glutamine Val: Valine Asp: Aspartic acid His: Histidine Met: Methionine Arg: Arginine Trp: Tryptophane Glu: Glutamic acid Ile: Isoleucine Asn: Asparagine Ser: Serine Tyr: Tyrosisne DNAmRNAProtein Transcription Different form of the same message DNA makes single stranded RNA (U replaces T) RNA leaves nucleus Translation Translate from nucleic acid language to amino acid language Uses codons, 3-base “word” that codes for specific a.a. “code” for an amino acid Several codons make a “sentence” that translates to a polypeptide (protein) The Genetic Code Am. Biochemist Marshall Nirenberg began to crack the genetic code in the 1960s Built RNA model with uracil, called poly U, conducted experiments with it and figured out UUU coded for amino acid phenylalanine Scientists used his procedures to figure out the other amino acids represented by codons Stop codons: UAA, UGA, UAG SIGNAL END OF GENETIC MESSAGE Start codon: AUG SIGNAL TO START TRANSLATING an RNA transcript Start Codons Stop Codons AUG UAA UGA UAG Three Types of RNA mRNA tRNA rRNA Three Types of RNA… #1 mRNA (messanger RNA) RNA transcribed from DNA template RNA polymerase (enzyme) links RNA nucleotides together Modified in nucleus before if exits RNA splicing: process in which Introns are removed and exons re joined together to make a continuous coding mRNA molecule Introns Internal non-coding regions of DNA and mRNA Space fillers They are cut out of mRNA before it is allowed to leave the nucleus Process is called RNA splicing (processing) Exons Coding region of DNA and mRNA that will be translated (Expressed) VERY important part of mRNA…it is carrying the message from DNA (def can’t cut this out) Three Types of RNA…#2 tRNA (transfer RNA) The interpreter Translate 3-letter base codes into amino acids Carries anti-codon on one end (three letters opposite of what is on mRNA) Carries amino acid on other end Anti-codon recognizes codon and attaches Three Types of RNA…#3 rRNA (ribosomal RNA) Found in ribosome Ribosome composed of 2 subunits: Small subunit for mRNA to attach Large Subunit for two tRNAs to attach “P” site: holds the tRNA carrying the growing polypeptide chain “A” site: holds the tRNA that is carrying the next a.a. to be added to the chain When stop codon (UAA, UAG, UGA) is reached, translation ends and polypeptide is released from tRNA by hydrolysis DNA and Sickle Cell Anemia Review hemoglobin 2 alpha chains 2 beta chain Sickle Cell Anemia Inherited blood disorder SUBSTITUTION Mutation to DNA sequence that codes for beta chains One DNA nucleotide is replaced with a different nucleotide Normal amino acid sequence for beta chain: VAL-HIS-LEU-THR-PRO-GLU-GLU-LYS HbA allele (normal hemoglobin allele) DNA triplet code for Glu is CTT Mutated amino acid sequence for beta chain: VAL-HIS-LEU-THR-PRO-VAL-GLU-LYS HbS allele (sickle cell allele) DNA triplet code for CTT is changed to CAT, which no longer codes for Glu, but instead Valine Effects of Sickle Cell Mutation Glutamic acid Found on outside of hemoglobin Hydrophilic aa Interacts with water molecules, makes hemoglobin soluble (good!) Valine Hydrophobic aa Does not interact with water, makes hemoglobin less soluble (BAD!) When abnormal hemoglobin is in area of LOW oxygen concentration, they stick together because the outside is now hydrophobic Abnormal hemoglobins form long chains of insoluble fibers These pull the red blood cells that contain the abnormal hemoglobin inwards and out of shape (become sickle shaped instead of round) Sickled RBC cannot move easily through blood Get stuck in capillaries Possibly fatal