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Honors Biology Ch. 12 Molecular Genetics CH. 12 Molecular Genetics I.DNA: The Genetic Material - DNA: The Chemical Basis of Heredity that forms the universal genetic code of cells A. Discovery of the Genetic Material - In the early 1900’s scientists were debating what was the genetic material: DNA vs. Protein. 1. Griffith - In 1928 Griffith showed that a heat killed, but lethal strain of pneumonia bacteria could ‘transform’ a harmless strain of pneumonia. Can the Genetic Trait of Pathogenicity Be Transferred between Bacteria? EXPERIMENT Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below: Living S (control) cells Living R Heat-killed (control) cells (control) S cells Mixture of heat-killed S cells and living R cells RESULTS Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells are found in blood sample. CONCLUSION Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells. 2. Avery - In 1944 Avery isolated DNA, proteins, and lipids from the heatkilled, lethal bacteria, and concluded that DNA was the transforming agent. 3. Hershey and Chase - In 1952 Hershey and Chase used bacteria-infecting viruses containing either radioactively label S or P to show that DNA was the genetic material. Phage head Viruses Infecting a Bacterial Cell DNA Bacterial cell 100 nm Tail Tail fiber Is DNA or Protein the Genetic Material of Phage T2? EXPERIMENT In their famous 1952 experiment, Alfred Hershey and Martha Chase used radioactive sulfur and phosphorus to trace the fates of the protein and DNA, respectively, of T2 phages that infected bacterial cells. 1 2 Agitated in a blender to 3 Mixed radioactively separate phages outside labeled phages with the bacteria from the bacteria. The phages bacterial cells. infected the bacterial cells. Phage Centrifuged the mixture 4 Measured the so that bacteria formed radioactivity in a pellet at the bottom of the pellet and the test tube. the liquid Radioactivity (phage protein) in liquid Radioactive Empty protein protein shell Bacterial cell Batch 1: Phages were grown with radioactive sulfur (35S), which was incorporated into phage protein (pink). Batch 2: Phages were grown with radioactive phosphorus (32P), which was incorporated into phage DNA (blue). DNA Phage DNA Centrifuge Radioactive DNA Pellet (bacterial cells and contents) Centrifuge Pellet Radioactivity (phage DNA) in pellet RESULTS Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells. When cultured, bacterial cells with radioactive phage DNA released new phages with some radioactive phosphorus. CONCLUSION Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material. B.DNA Structure - The structure of DNA was discovered by James Watson and Francis Crick in 1953. Watson and Crick with Their DNA Model - Rosalind Franklin used X-Ray diffraction to discover the shape and dimensions of DNA. Rosalind Franklin Franklin’s X-ray diffraction Photograph of DNA 1.Components of DNA (3 Main Parts) a. Sugar (Deoxyribose) b. Phosphate c. Bases c. Bases (1) Adenine (A) Guanine (G) (2) Cytosine (C) Thymine (T) 2. Nucleotide: - a subunit of a nucleic acid containing a sugar, a phosphate, and a base 3.DNA Shape: - double helix a. backbone - sugars and phosphates b. paired bases form on the inside c. Base Pairing Rule: A : T , C : G Link to an interview with James Watson (1:42) The Watson-Crick Model of DNA Structure II. Replication of DNA : - process by which DNA makes an exact copy of itself - occurs before mitosis during interphase A.Semiconservative Replication - DNA strands separate and serve as templates for rebuilding the other half. T A T A T A C G C G C T A T A T A A T A T A T G C G C G C G A T A T A T C G C G C G T A T A T A T A T A T C G C G C A G Free Nucleotides DNA Replication New double helix with 1 old & 1 new strand Parental DNA double helix 1. Unwinding - DNA helicase unwinds and unzips DNA molecule. - RNA primase adds a short segment (RNA primer) on each DNA strand. DNA Replication Overall direction of replication 1 Helicase unwinds the parental double helix. 2 Molecules of singlestrand binding protein stabilize the unwound template strands. 3 The leading strand is synthesized continuously in the 5 3 direction by DNA pol III. Leading strand Origin of replication Lagging strand Lagging strand Leading strand OVERVIEW DNA pol III Leading strand 5 3 Parental DNA Replication fork Primase DNA pol III Primer 4 Primase begins synthesis of RNA primer for fifth Okazaki fragment. 5 DNA pol III is completing synthesis of the fourth fragment, when it reaches the RNA primer on the third fragment, it will dissociate, move to the replication fork, and add DNA nucleotides to the 3 end of the fifth fragment primer. 4 DNA ligase DNA pol I Lagging strand 3 6 DNA pol I removes the primer from the 5 end of the second fragment, replacing it with DNA nucleotides that it adds one by one to the 3’ end of the third fragment. The replacement of the last RNA nucleotide with DNA leaves the sugarphosphate backbone with a free 3 end. 2 1 3 5 7 DNA ligase bonds the 3 end of the second fragment to the 5 end of the first fragment. 2. Base Pairing - DNA polymerase adds nucleotides with the complementary base on the 3’ end of each strand. - The leading strand is synthesized continuously by adding nucleotides to 3’ end. - The lagging strand is synthesized from Okazaki fragments which are later connected by DNA ligase. 3. Joining - RNA primers are replaced by DNA nucleotides - DNA ligase connects the fragments together. Link to DNA Replication Video Clip (2:19) A Summary of DNA Replication Overall direction of replication Lagging Leading strand Origin of replication strand 1 Helicase unwinds the parental double helix. 2 Molecules of single- 3 The leading strand is strand binding protein synthesized continuously in the stabilize the unwound 5 3 direction by DNA pol III. template strands. DNA pol III Lagging strand OVERVIEW Leading strand Leading strand 5 3 Parental DNA 4 Primase begins synthesis of RNA primer for fifth Okazaki fragment. 5 DNA pol III is completing synthesis of the fourth fragment, when it reaches the RNA primer on the third fragment, it will dissociate, move to the replication fork, and add DNA nucleotides to the 3 end of the fifth fragment primer. Replication fork Primase DNA pol III Primer 4 DNA ligase DNA pol I Lagging strand 3 6 DNA pol I removes the primer from the 5 end of the second fragment, replacing it with DNA nucleotides that it adds one by one to the 3’ end of the third fragment. The replacement of the last RNA nucleotide with DNA leaves the sugarphosphate backbone with a free 3 end. 2 1 3 5 7 DNA ligase bonds the 3 end of the second fragment to the 5 end of the first fragment. Telomeres 1 µm B. DNA Replication in Prokaryotes - Prokaryotic chromosomes made of one circular DNA strand without proteins. - DNA replication begins at a single origin of replication and occurs very quickly. Origins of Replication in Eukaryotes Origin of replication 1 Replication begins at specific sites where the two parental strands separate and form replication bubbles. Bubble Parental (template) strand Daughter (new) strand 0.25 µm Replication fork 2 The bubbles expand laterally, as DNA replication proceeds in both directions. 3 Eventually, the replication bubbles fuse, and synthesis of the daughter strands is complete. Two daughter DNA molecules (a) In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome. (b) In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). C. Chromosome Structure - Chromosomes of eukaryotes are made of DNA and proteins forming bead-shaped nucleosomes which are coiled and folded to form chromatin. Structure of a Eukaryotic Chromosome C. Chromosome Structure - Chromosomes of eukaryotes are made of DNA and proteins forming bead-shaped nucleosomes which are coiled and folded to form chromatin. Link to DNA Packaging Video Clip (1:43) III.DNA, RNA, and Protein A. ‘Central Dogma’ - DNA codes for RNA which guides protein synthesis. 1. Gene - a specific sequence of bases in DNA that determines the sequence of amino acids in a protein 2. Proteins - composed of 50-20,000 amino acids - 4 Levels of Structure: a) Primary: - sequence of amino acids b) Secondary: - either -helical or “β-pleated sheet” c) Tertiary: - globular (3-dimensional) d) Quaternary: - 2+ polypeptides combined Illustration of Protein Structure Primary (Amino Acid Sequence) Tertiary (Bending) Quaternary (Layering) Secondary (Helix) B. RNA Structure: - Nucleic acid that makes protein B.RNA Structure: Shape Sugar Base Size Location Function DNA double helix deoxyribose thymine very large nucleus - stores genetic info - replication - makes RNA RNA single helix ribose uracil smaller cytoplasm - makes protein C.Transcription: - the copying of a genetic message from DNA to RNA Original DNA C.Transcription: - the copying of a genetic message from DNA to RNA DNA base pairs separate C.Transcription: - the copying of a genetic message from DNA to RNA DNA half ‘transcribes’ RNA C.Transcription: - the copying of a genetic message from DNA to RNA Link to Transcription Video Clip (1:54) RNA released to make protein Transcription: First Two Steps Transcription: Last Step Three Types of RNA mRNA A G A U G C G A G U U A U G G codons Ribosome contains rRNA Met Amino acid tRNA anticodon Large subunit 1 2 Small subunit tRNA docking sites UGA D. Messenger RNA (mRNA): - carries the information for making a protein from DNA to the ribosomes - acts as a template (pattern) - contains codons: triplets of bases that code for a particular amino acid - Start Codon: (AUG) - marks the start of a polypeptide - Stop Codon: (UAA, UAG, UGA) - marks the end E. Transfer RNA (tRNA): - carries amino acid to specific place on mRNA - contains Anticodon: triplet of bases complimentary to mRNA codon F. Ribosomal RNA (rRNA): - combined with protein into ribosomes (site of protein synthesis) IV. Translation: - protein synthesis - decoding the "message" of mRNA into a protein Link to Translation Video Clip (2:05) Information Flow: DNA RNA Protein Translation: Initiation Translation: Elongation 1 Translation: Elongation 2 Translation: Elongation 3 Translation: Elongation 4 Translation: Elongation 5 Translation: Termination V. Genetic Regulation and Mutations A. Prokaryotic Gene Expression - Prokaryotic cells regulated gene expression with a set of genes called an operon. - An operon consists of 1. Operator 2. Promoter 3. Regulatory gene 4. Protein coding genes Trp Operon: Inducible Operon Regulation of a Metabolic Pathway (a) Regulation of enzyme activity Precursor Feedback inhibition (b) Regulation of enzyme production Enzyme 1 trpE Gene Enzyme 2 trpD Gene Enzyme 3 Regulation of gene expression trpC Gene – Enzyme 4 trpB Gene – Enzyme 5 Tryptophan trpA Gene The trp operon: regulated synthesis of repressible enzymes trp operon Promoter Promoter Genes of operon trpD trpC trpE trpR DNA trpB trpA Operator Regulatory gene mRNA 3 RNA polymerase Start codon Stop codon mRNA 5 5 E Protein Inactive repressor D C B A Polypeptides that make up enzymes for tryptophan synthesis Tryptophan absent, repressor inactive, operon on. RNA polymerase attaches to the DNA at the promoter and transcribes the operon’s genes. DNA No RNA made mRNA Active repressor Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off. As tryptophan accumulates, it inhibits its own production by activating the repressor protein. B. Eukaryotic Gene Expression - Eukaryotic cells regulate gene expression using various transcription factors and other processes. Eukaryotic Gene Expression Distal control element Activators Enhancer 1 Activator proteins bind to distal control elements grouped as an enhancer in the DNA. This enhancer has three binding sites. Promoter Gene TATA box General transcription factors DNA-bending protein 2 A DNA-bending protein brings the bound activators closer to the promoter. Other transcription factors, mediator proteins, and RNA polymerase are nearby. Group of Mediator proteins RNA Polymerase II Chromatin changes 3 The activators bind to certain general transcription factors and mediator proteins, helping them form an active transcription initiation complex on the promoter. Transcription RNA processing mRNA degradation RNA Polymerase II Translation Protein processing and degradation Transcription Initiation complex RNA synthesis C. Mutations - any change in the nucleotide sequence of DNA - usually recessive - many are harmful - some are neutral - some beneficial (leads to evolution) - Mutagens: cause mutations 1) Radiation- UV, X-rays, etc. 2) Chemicals (asbestos, etc.) D. Types of Mutations: 1. Point Mutation - change of a single base - ex: sickle-cell anemia AUG GGG CUU CUU AAU AUG GGG CAU CUU AAU Normal Red Blood Cells Sickled Cells Link to a Video Clip about Sickle Cell Disease(0:59) 2. Frameshift Mutation - addition or deletion of a single base AUG GGG CUU CUU AAU AUG GGG CAU UCU UAA U 3. Chromosomal Mutation - change in an entire chromosome or in chromosome number within a cell a) Translocation: Normal Translocation b) Inversion: Normal Inversion c) Insertion: Normal Insertion d) Deletion : Normal Deletion The End (Cytoplasm) DNA (Nucleus) 1 Transcription rRNA mRNA tRNA + Proteins tRNA Ribosomes mRNA tRNA-AA 2 Translation Inactive Protein Active Protein 3 Modification 4 Degradation Substrate Product Amino Acids Overview of Information Flow Complementary Base Pairing gene G C A T G G G A G T template DNA strand T (a) complementary DNA strand C G T A C C C T C A A codons (b) mRNA G C A U G G G A G U U anticodons (c) tRNA U A C C C U C A amino acids (d) protein Methionine Glycine Valine A Incorporation of a Nucleotide into a DNA Strand New strand Template strand Sugar A Base Phosphate 3’ end 5’ end 3’ end 5’ end T A T C G C G G C G C A T A P OH P Pyrophosphate 3’ end C C OH Nucleoside triphosphate 2 P 5’ end 5’ end Structural Proteins Horn Hair Spiderweb Hair Structure Hair Cell Single hair Microfibril Protofibril | S | Hydrogen bonds S | | S | S | disulfide bridges Curling of Hair S | | | S | | S S | | | S | S S | | | S | Permanent Wave S | Naturally Curly Hair | S | S | | S Straight Hair LUX Operon Controls Light Production Light Emitted by Bacterial Luciferase Video Clip about how a single transcription factor controls the LUX operon, which contains five genes necessary to produce bioluminescence in bacteria (2:26).