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DNA: The Molecule of Inheritance Mendel’s experiments proved that molecules from the parents were transferred to offspring These molecules could store genetic info, be replicated, and undergo mutations But what type of molecule? Could it be a lipid, protein, nucleic acid, carbohydrate? Scientists narrowed it down to 2 possibilities: nucleic acids or proteins What did they look like? What was their structure? Early DNA Experiments: Griffith Inject mice with live R bacteriamice live, no live R cells in blood Inject mice with live S bacteriamice die, live S cells in blood Inject mice with dead S bacteriamice live, no live S cells in blood Inject mice with live R bacteria + dead S bacteriamice die, live S cells in blood What happened in the last experiment? Early Experiments: Griffith Interpretation of results Heat killed S cells, but not hereditary material This material was transferred to R cells transforming them into S cells Transformation Permanent change, after 100s of generations transformed R cells still contained the instructions on causing infection Early Experiments: Avery (1944) Early Experiments: Avery Interpretation of results If add protein digesting enzymes, protein destroyed, but DNA intactR cells transformed into S cells, mice die If add DNA digesting enzymes, DNA destroyed, protein intactR cells did not transform, mice live Early Experiments: Hershey and Chase (1950s) • Used T4 bacteriophage- bacterial virus • Made of a outer protein coat which protects its inner genetic material • In order to infect its hosts its genetic material must be introduced into the host first • Was it the protein coat or the inner genetic material that caused infection? They knew that 1) T2 phages were 50:50 Protein:DNA, 2) Viral reproduction occurs inside bacterial cell Early Experiments: Hershey and Chase (1950s) Radiolabel protein coat with 35S, after T4 infection of bacteria found label outside Radiolabel inner material with 32P, after T4 infection of bacteria found label inside Early Experiments: Hershey and Chase Interpretation of results Infection of bacteria cells is caused by the transfer of it genetic material to the inside Whichever molecule was found on inside must be molecule of inheritance Proved beyond any doubt that DNA was the molecule of inheritance But what did it look like? Discovery of the Structure of DNA DNA consisted of only 4 nucleotides: Adenine, Thymine, Cytosine, Guanine deoxyribose sugar, phosphate group, base T, C pyrimidines (one ring) A, G purines ( 2 rings) Discovery of the Structure of DNA Chargaff’s Laws (1949): A=T, G=C, DNA was species specific Structure solved; it was a helix Discovery of the Structure of DNA: X-Ray Diffraction (Wilkins, Franklin) X-ray diffraction of DNA fibers gave a regular repeating pattern of atoms in the DNA Discovery of the Structure of DNA: Watson and Crick Experiments (1953) • Using all previous data from other scientists and experiments, Watson and Crick published a 1 page paper on solving the structure of DNA • Watson, Crick, Wilkins received Nobel Prize in 1962 Structure of DNA 2 nm in diameter Distance between each base pair= 0.34nm Double stranded helix, “twisted ladder” A base pairs with T, C base pairs with G, 2 H bonds for A-T, 3 H bonds for C-G One strand runs 5’3’ (right side up), the other strand runs 3’5’ (upside down) DNA Replication • Duplication of DNA. When does it occur? • Semiconservative-part of the original DNA molecule is conserved during replication. • 1 DNA molecule2 DNA molecules each consisting of a new and old strand • New strand is made using old strand as template and base pair rules DNA Replication What would be the complementary strand of the following DNA sequence? ACGTATACGTGC The following piece of DNA is to be replicated. Give the correct daughter strand. TTACCGGTTC DNA Replication and Complementary Strands ACGTATACGTGC original TGCATATGCACG complementary TTACCGGTTC original AATGGCCAAG replicated DNA Replication: Enzymes Involved • Topoisomerase and helicase-unwind and uncoil DNA (break H bonds) • DNA polymerase adds the correct nucleotide in the 5’-3’ direction only • DNA pol moves in the forward direction on one strand, moves in the reverse direction on the other strand, however, always moving in the 5’-3’ direction DNA Replication • Replication is continuous on leading strand; discontinuous on the lagging strand • DNA pol only adds 5’3’ • Lagging strand is composed of Okazaki fragments that must be linked together by ligase Prokaryotic vs Eukaryotic Replication • Bacteria have a circular chromosome replication occurs in 2 directions at the origin of replication • Replication is fast minutes • Eukaryotes have long, linear chromosomes replication begins at many locations replication bubbles, replication forks • Replication is slower hours DNA Repair • What happens if the incorrect base is added and a • • • • mismatch base pair occurs? Ex. A-G or C-T DNA Pol has a proofreading function It will cut out the incorrect base and put back the correct base Ligase comes in and repairs the “cut” in the DNA Results in a very low error rate 1 out of 100 million base pairs From DNA to Proteins DNA is the genetic instructions for life, but DNA itself does not do work to sustain life. Which type of molecule is responsible for all of a cell’s processes? Flow of genetic information instructions translator worker DNA RNA Proteins transcription translation nucleus cytoplasm Where does transcription take place in a prokaryotic cell? Comparison of DNA to RNA RNA Single stranded Ribose sugar Bases A, Uracil, C, G A-U, C-G base pairs 3 types/functions Unstable, easily degrades DNA Double stranded Deoxyribose sugar Bases A, T, C, G A-T, C-G base pairs 1 type/function Very stable over time Types of RNAs Messenger RNA (mRNA)-carries the protein building instructions: 1 gene=1 mRNA=1 protein Ribosomal RNA (rRNA)-this RNA along with other proteins make up ribosomes (site of protein synthesis) Transfer RNA (tRNA)-brings correct amino acid to the ribosome; binds to mRNA sequence The Genetic Code DNARNAAmino Acids (proteins) How many different nucleotides are there? How many different amino acids are there? How would the nucleotides specify each of the amino acids? The Genetic Code Solved! There are only 4 nucleotides to specify 20 different amino 41 42 43 acids If use 1 nucleotide per amino acid, only can specify 4 Using 2 nucleotides, only can specify 16 If use 3 nucleotides per amino acid, can specify 64 amino acids 3 nucleotides = 1 codon, 1 codon per amino acid The Genetic Code The code is universal The code is degenerative most amino acids have more than 1 codon. Why? Each codon has only 1 meaning, CCA is different than CUA, and can specify a different amino acid Has 1 start signal and 3 stop signals Transcription: DNARNA • Initiation of Transcription • RNA pol binds to specific DNA • • • • sequence called a promoter which is usually at the beginning of a gene DNA unwinds using enzymes RNA pol adds nucleotides in 5’3’ direction: A-U, C-G RNA pol falls off at end of gene, releasing mRNA transcript Many RNA pols work simultaneously to produce mRNAs RNA Transcript Processing and Modification • Newly synthesized • • • • • transcripts are not ready to make a protein 3 Modifications are made: Introns are cut out by “Splicing” Genes exons and introns Exons coding sequences that make a protein Introns noncoding (junk) sequences RNA Transcript Processing and Modification 5’ cap is added Provides an docking area for the ribosome to bind during translation Prevents degradation 3’ Poly A (adenine) Tail is added 100-200 As added to prevent degradation by enzymes Similarities Between Replication and Transcription Occur in the nucleus Use DNA as template to build new strands Add nucleotides in the 5’3’ direction Add nucleotides according to base pair rules: A-T,U or C-G Both use helicase and topoisomerase to unwind DNA Differences Between Replication and Transcription Replication DNADNA copy Copy all of the DNA Copy is double stranded Use DNA pol Use A-T, C-G Transcription DNARNA copy Copy only part of DNA Copy is single stranded Use RNA pol Use A-U, C-G Transcription: DNARNA The following sequence is to be transcribed: AATCGGTCGATGG What is the sequence of the transcript? AATCGGTCGATGG DNA UUAGCCAGCUACC RNA Translation: RNAProteins Occurs in the cytoplasm, inside of ribosomes (attached to ER) Requires a mRNA with a start/stop codon Requires a tRNA Requires amino acids Translation: RNAProteins Role of tRNA • tRNA-2 Binding sites • mRNA binding site called the anticodon • UAA CGC AAC mRNA • AUU GCG UUG tRNA • amino acid binding site, to bring the correct amino acid to growing polypeptide chain Translation: Role of Ribosomes • Site of protein synthesis • Composed of large and small subunits • During translation: • Small subunit binds mRNA, then combines with large subunit to form intact ribosome (initiation) • When small and large subunits combine, form E site (exit), P site (peptide), and an A site (amino acid) Translation Initiation: initiator tRNA + small ribosome unit, mRNA + large ribosome unit Elongation: initiator tRNA (start codon) P site Where does the 2nd tRNA add? Translation: RNAProteins 2nd tRNA A site peptide bond 1st + 2nd aa Initiator tRNA released E site 2nd tRNA P site 3rd tRNA A site Translation: Elongation Peptide bond 2nd + 3rd aa This is repeated many times until a stop codon is reached Proteins can have as little as 30 aa or up to 1000 aa 12-17 aa are added every second!!!! Translation • Termination: stop codon is reached, mRNA released, and ribosome subunits separate, polypeptide chain is released • What happens to the newly synthesized protein? • Golgi for processing and shipping by exocytosis • Used in the cell it was made Reading the Genetic Code • There are 64 codons (61 specify amino acids, 3 are stops) • Some amino acids have more than one codon, ex. arginine, leucine • For codon GAC, first find the first base on code, second, and finally third; where all lines intersect that is the amino acid specified. GAC = aspartate Genetic Code Problem What would be the amino acid sequence specified by the following DNA sequence? TAC GCT ATA CCC ATT How many amino acids would be made? Genetic Code Problem TAC GCT ATA CCC ATT DNA AUG CGA UAU GGG UAA RNA start-arginine-tyrosine-glycine-stop amino acid (methionine) Structure of Eukaryotic Chromosomes Chromosomes (made of DNA) have proteins that help tightly package DNA in the nucleus Histones They arrange the DNA around “beads” called nucleosomes heterochromatin