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DNA C G T A Base pair C Hydrogen bond T A T G C G A T A C G C G T T C A G A A T A T A G A Ribbon model T C T Partial chemical structure Computer model What does the cell use DNA for? • Gives you traits What IS a trait? • Physical structure produced by a protein! • DNA controls the production of proteins. What do we know about making proteins? DNA is in the NUCLEUS RIBOSOMES, ER, and GOLGI are in the CYTOPLASM How does that work? • RNA acts as a messenger to carry information from the DNA in the nucleus to the ribosomes in the cytoplasm What’s RNA? • Nucleic Acid • Similar to DNA • Some differences Nitrogenous base (A, G, C, or U) Phosphate group Uracil (U) Sugar (ribose) What’s RNA? FEATURE DNA RNA Subunits Nucleotide Nucleotide Strands 2 – double helix 1 (mostly) Sugar Deoxyribose Ribose Bases A = T; C = G A = U; C = G Protein Synthesis Overview DNA is located in the NUCLEUS Protein Synthesis Overview A messenger RNA (mRNA) copy is made of DNA. Protein Synthesis Overview mRNA leaves the nucleus and goes to the ribosome Protein Synthesis Overview Ribosome uses mRNA to assemble amino acids in the correct order to make a specific protein Genes to Polypeptides • Polypeptides = chains of AA = proteins • 20 different AA exist • specific polypeptide has specific AA sequence • Sequence of AA determines the shape and function of a protein Genes to Polypeptides • Sequence of bases in DNA determine AA sequence • “Genes” store order of AA in a code in DNA • One specific gene will yield one* specific polypeptide – polypeptide = protein that does a job! DNA & Genetic Code • There are 20 amino acids, and a stop • How can DNA specify 21 things with only four bases? Genetic Code G A T C 4 • IF: 1 base = 1 amino acid • THEN: how many amino acid possibilities are there? Genetic Code G A T C G A T C 4 x 4 = 16 • IF: 2 bases = 1 amino acid • THEN: how many amino acid possibilities are there? Genetic Code G A T C G A T C 4 x 4 x 4 = G A T C 64 • IF: 3 bases = 1 amino acid • THEN: how many amino acid possibilities are there? DNA & Genetic Code • In a gene, every three bases code for a specific amino acid (one of the 20) • 4 x 4 x 4 = 64 total possiblities • One amino acid can be coded for by more than one triplet DNA & Genetic Code Genetic code is composed of codons made up of of base triplets DNA & Genetic Code • The genetic code is both universal and degenerate. – Universal = found in all living organisms – Degenerate = having more than one base triplet (codon) to code for one amino acid Protein Synthesis Overview • DNA is located in the nucleus Protein Synthesis Overview • Ribosomes are located in the cytoplasm Protein Synthesis Overview • Messenger RNA carries “message” from DNA to ribosomes Transcription • Genes are made of DNA • DNA cannot leave the nucleus • A copy must be “transcribed” into RNA • RNA exits nucleus http://www.fed.cuhk.edu.hk/~jo hnson/teaching/genetics/anima tions/transcription.htm Transcription 1. INITIATION: RNA polymerase uncoils DNA double helix 2. ELONGATION: RNA polymerase creates a new mRNA strand using free RNA nucleotides; a single DNA template strand is used Transcription 3. RNA nucleotides attached together (type of reaction?) via RNA polymerase 4. TERMINATION: New mRNA strands separates from DNA 5. DNA reforms Animation: Campbell Ch 10 – 10_9 Transcription GENE Contains the instructions to assemble one protein TRANSCRIPTION The process by which an mRNA copy is made of a DNA sequence RNA POLYMERASE Enzyme that catalyzes synthesis of mRNA strand PROMOTER REGION Sequence of DNA bases within gene where RNA polymerase binds CODING REGION Sequence of DNA bases that codes for the actual structure of the protein TERMINATOR REGION Sequence of DNA were RNA polymerase stops transcribing. CODON 3 bases of DNA or RNA; specifies 1 amino acid What’s it look like? So, the new mRNA strand was just made, now what? Final Steps – Eukaryotes ONLY 1. mRNA Splicing – – – INTRONS: non-coding regions of the mRNA strand EXONS: coding regions of the mRNA strand Introns are spliced out of final mRNA Final Steps – Eukaryotes ONLY 2. 5’ Cap – – Modification to 5’ end of mRNA Ensures stability of mRNA Final Steps – Eukaryotes ONLY 3. 3’ poly-A tail – – Addition of poly-A to 3’ end of mRNA Protects RNA from nucleases Exon Intron Exon Intron Exon DNA Cap RNA transcript with cap and tail Transcription Addition of cap and tail Introns removed Tail Exons spliced together mRNA Coding sequence Nucleus Animation: Cain Ch13a03 - Transcription Cytoplasm Transcription in a cell • Multiple genes can be transcribed at the same time • The same gene can be transcribed at the same time Translation Nucleus Ribosome (cytoplasm) DNA mRNA Protein Transcription Translation Where we are now. Translation Summary • Instructions in the mRNA are used by a ribosome to assemble amino acids in the correct order • Order of amino acids gives the protein its shape • Shape gives protein its function Translation Summary The Key Players • mRNA • tRNA • rRNA Animation: Cain Ch13a07 - Translation The Stages of Translation • Initiation • Elongation • termination mRNA (messenger RNA) • copy of the directions to make the product (protein) • tells the ribosome the correct sequence of Amino Acids while putting together the protein • each codon (3 bases) directs a specific amino acid to be added to the growing protein tRNA (transfer RNA) • the delivery RNA; delivers specific Amino Acids to the ribosome • composed of RNA • anticodon binds to a corresponding codon on mRNA • Carries one specific amino 6.4.1 Translation rRNA (ribosomal RNA) • ribosomes are made of RNA and protein • composed of two subunits: the 30s and 50s subunits • two tRNA binding sites; one mRNA binding site Translation INITIATION • small (30S) ribosome subunit binds to mRNA at the 5’ end of the mRNA • 30S moves along mRNA 5’ to 3’ until it hits the start codon AUG Translation INITIATION • large subunit binds • Methionine tRNA moves into ribosome Translation INITIATION • another tRNA, with the anticodon complementary to the next codon binds to the ribosome Translation ELONGATION • first amino acid added • ribosome moves down mRNA to next codon • next tRNA comes in • its amino acid is bound to the polypeptide chain • ribosome moves down mRNA to next codon Translation TERMINATION • the ribosome encounters a stop codon • no tRNA molecule has an anticodon for this codon Translation TERMINATION • polypeptide is released and ribosome disassociates Animation: Campbell Ch 10 – 10_14 Translation Where does translation happen? • Cytoplasmic (free) Ribosomes proteins for use in cytoplasm • Rough ER (attached) Ribosomes proteins secreted or used in lysosomes DNA molecule Gene 1 • DNA Gene 2 – TRANSCRIPTION • RNA Gene 3 – TRANSLATION • PROTEIN DNA strand A A A C C G G C A A A A U U U G G C C G U U U U Transcription RNA Codon Translation Polypeptide Amino acid Interpreting the Genetic Code Strand to be transcribed Second base T A C T T C A A A A T UUU DNA A T G A A G T T T T A G C U C U UUC UUA UUG Phe Leu CUU Transcription C A U G A A G U U U U A RNA G A CUC CUA Stop codon Translation Lys Phe UCA Ser UAC U C CCU CAU CGU CCC CCA Pro CAC CAA AUC lle ACC AUA ACA GUA UGC Cys UAG Stop AAU Val UGU UCG ACU GUC Tyr UGA Stop A UGG Trp G AUU Met or start G UAA Stop CAG GUG Polypeptide Met UCC CCG GUU G UAU UCU CUG AUG Start codon Leu A Thr AAC AAA ACG AAG GCU GAU GCC GCA GCG Ala GAC GAA GAG His Gln Asn Lys Asp Glu CGU CGA U Arg G CGG AGU AGC AGA AGG C A Ser Arg U C A G U GGC C Gly GGA A GGG G GGU Animation: Starr Ch 14 – Genetic code Changes in the Genetic Code • MUTATION = change in the nucleotide sequence of DNA Normal hemoglobin DNA Mutant hemoglobin DNA C T T mRNA C A T G U A mRNA G A A Normal hemoglobin Glu Sickle-cell hemoglobin Val Changes in the Genetic Code • MUTATION = change in the nucleotide sequence of DNA Normal hemoglobin DNA Mutant hemoglobin DNA C T T mRNA C A T G U A mRNA G A A Normal hemoglobin Glu Sickle-cell hemoglobin Val Changes in the Genetic Code • MUTATION = change in the nucleotide sequence of DNA Normal hemoglobin DNA Mutant hemoglobin DNA C T T mRNA C A T G U A mRNA G A A Normal hemoglobin Glu Sickle-cell hemoglobin Val What causes mutations? • Spontaneous mutations: uncorrected errors in replication What causes mutations? • Spontaneous mutations: uncorrected errors in replication • Harmful environmental agents: UV light, radiation, chemicals Radiation damages DNA Radiation as a cancer treatment Why would radiation be a treatment for cancer? • What type of cells would radiation affect most: rapidly dividing or rarely dividing cells? • Cancer cells are very rapidly dividing cells • Radiation targets ALL rapidly dividing cells, not just cancer cells What causes mutations? • Spontaneous mutations: uncorrected errors in replication • Harmful environmental agents: UV light, radiation, chemicals • Transposable elements: “jumping genes” Mutations • Sickle Cell Anemia – Single-base substitution Sickle Cell Anemia Missense mutation = single base substitution Changes one amino acid Variable effect on protein depending on how much structure is changed Missense Mutations • Tay Sachs Disease – Single-base substitution in HexA gene Missense Mutations • Cystic Fibrosis – Single-base substitution in CFTR gene Nonsense mutation = single base substitution that introduces a STOP Truncates protein Often more severe Nonsense Mutations • Cystic Fibrosis – More severe form • Duchenne Muscular Dystrophy – Dystrophin – connects cytoskeleton to extracellular matrix Silent mutation = single base substitution that doesn’t change an amino acid Second base C U UUU U UUC UUA UUG Phe Leu CUU C A CUC CUA Leu UAU UCU UCC UCA Ser UAC CAU CGU CCC CCA Pro CAC CAA AAU AUC lle ACC AUA ACA GUA GUG U C CCU ACU Val UGC Cys UAG Stop AUU GUC UGU UCG CAG GUU Tyr UGA Stop A UGG Trp G CCG Met or start G UAA Stop CUG AUG G A Thr AAC AAA ACG AAG GCU GAU GCC GCA GCG Ala GAC GAA GAG His Gln Asn Lys Asp Glu CGU CGA U Arg G CGG AGU AGC AGA AGG C A Ser Arg U C A G U GGC C Gly GGA A GGG G GGU Insertion mutation = addition of one or more nucleotides Can change entire protein after mutation Deletion mutation = deletion of one or more nucleotides Can change entire protein after mutation Frameshift mutation = Change of “reading frame” in DNA Can change entire protein after mutation Insertion or deletion Cancer • BRCA1 - increased risk of developing breast cancer • Mutations due to mutagens Summary of Mutations • Base Substitution – Missense – Nonsense – Silent • Frameshift – Insertion – Deletion • Which kind would be most likely to cause disease? Normal gene A U G A A G U U U G G C G C A mRNA Protein Met Lys Phe Gly Ala Base substitution A U G A A G U U U A G C G C A Met Lys Base deletion Phe Ser Ala U Missing A U G A A G U U G G C G C A U Met Lys Leu Ala His Good information about genes and mutations • http://ghr.nlm.nih.gov/handbook/basics Effect of Mutation on Protein Structure Effect of Mutation on Protein Structure Transcription Assembly of RNA on unwound regions of DNA molecule mRNA processing mRNA mature mRNA transcripts Translation At an intact ribosome, synthesis of a polypeptide chain at the binding sites for mRNA and tRNAs rRNA ribosomal subunits Convergence of RNAs tRNA mature tRNA cytoplasmic pools of amino acids, ribosomal subunits, and tRNAs Protein Animation: Starr Ch 14 - Protein Synthesis in Prokaryotes vs Eukaryotes In-class assignment – Protein Synthesis • Complete the classwork assignment Gene Expression • Every cell in your body came from 1 original egg and sperm • Every cell has the same DNA and the same genes 76 Gene Expression • Every cell in your body came from 1 original egg and sperm • Every cell has the same DNA and the same genes • Each cell is different, specialized • Differences due to gene expression – Which genes are turned on – When the genes are turned on – How much product they make 77 Genetic Potential • Embryonic Stem Cells – Can differentiate to become any type of cell in the body • Adult Stem Cells – Can differentiate to become several types of cell Genetic Potential • Plants in Cell Culture • Plants creating roots Root of carrot plant Single cell Root cells cultured in nutrient medium Cell division in culture Plantlet Adult plant Genome Size • Genome: total amount of DNA • Prokaryotes – 0.6 to 30 million base pairs – Approximately 2,000 genes • Eukaryotes – 12 million to 1 trillion base pairs – Humans have ~25,000 genes 80 Organization of DNA • Prokaryotes – Several million base pairs one circular piece – Related genes grouped together – Mostly coding DNA 81 Organization of DNA • Eukaryotes – Billions of base pairs – several linear chromosomes – Genes not grouped – Mostly non-coding DNA 82 Noncoding DNA • Spacer DNA • Transposons – “selfish DNA” 83 DNA Packaging • • • • Eukaryotic chromosomes are very large Must be packaged to fit inside nucleus Unavailable for transcription Unpacking must occur before transcription 84 Levels of Packaging • Chromosome – fully condensed • Tightly packed loops • 30 nm fibers • Histone spool • Double helix 85 Patterns of Gene Expression • Bacteria directly exposed to environment • Respond to changes in nutrient availability directly – Make enzymes for nutrients when they are present – Turn genes off when they are not 86 Patterns of Gene Expression • Eukaryotic cells • Tissue specific expression • Housekeeping genes 87 Gene Expression: Development • Embryo development depends on gene expression • Timing of expression vital • Controlled by cascades of gene expression 88 Levels of Gene Control 1. 2. 3. 4. 5. 6. 7. 89 Packaging Transcription mRNA maturation mRNA breakdown Translation Protein Regulation Protein Degradation 1. Packaging • If the DNA isn’t unwrapped from the histones, it can’t be transcribed • DNA Methylation • HistoneMethylation Animation: Campbell Ch 11 – DNA Packing 1. Packaging Methylation • DNA marked with a methyl group can be identified by an enzyme 1. Packaging • X chromosome Inactivation – Females have 2 X chromosomes – One gets methylated and inactivated during development 1. Packaging • “Copycat” – first cloned cat 1. Packaging • Agouti, mottled and yellow mice 1. Packaging • Methylation is required for development – Lethal to eliminate methylation in animals – Not lethal in plants, but profound effects on development 1. Packaging • Imprinting – methylate and silence genes on one parent’s chromosome specifically – About 1% of total human genes (about 300 genes) 1. Packaging • Parental DNA contributions to embryo are marked 1. Packaging • Normal = maternal expressed, paternal silenced • Prader-Willi = paternal allele lost, maternal allele present • Angelman = maternal allele lost, paternal allele present 1. Packaging • Cancer cells are often aberrantly methylated 1. Packaging • Cancer cells are often aberrantly methylated 2. Transcription • Control when and how much a gene is transcribed 2. Example of Transcriptional Control: The Lac Operon in Bacteria • E. coli lactose sugar utilization genes • When lactose is present, bacteria needs to have the proteins coded for by these genes – Lactase Enzymes Lac Operon Animation (online) 2. Example of Transcriptional Control: The Lac Operon in Bacteria • Operon: group of nucleotide sequences including an operator, a promoter, and one or more genes that are controlled as a unit to produce messenger RNA (mRNA) *The Operon model is one example of gene expression regulation 6.3.2 Lac Repressor Protein • NO LACTOSE: repressor binds to the DNA and prevents RNA pol from binding (no transcription) • No lactase is produced 6.3.2 Lac Repressor Protein • LACTOSE: repressor binds lactose and changes shape. Now repressor can’t bind DNA • Lactase is produced; lactose is metabolized 2. Example of Transcriptional Control: The Trp Operon Tryptophan AA genes ANIMATION (Ch14a03) 107 2. Transcriptional Control - Eukaryotic Gene Expression • • • • No operons More complex than prokaryotic Many different types of regulatory proteins Many DNA elements controlling each gene 108 2. Transcriptional Control - Eukaryotic Gene Expression • OFF: proteins are produced that bind to gene preventing RNA polymerase from binding 3. mRNA Maturation • If 3’ cap, 5’ poly-A tail are not added, mRNA cannot be transported out of the nucleus and used • mRNA can be “alternatively spliced” to generate different transcripts Exons DNA RNA transcript RNA splicing mRNA or 4. mRNA Breakdown • If mRNA is broken down more quickly, it can be used fewer times 5. Translational Regulation • Inhibit any of the steps of translation and the mRNA can’t be used 6. Protein Regulation • Activate or inactivate the newly made protein – Phosphorylation – Acetylation 6. Protein Regulation • Activate or inactivate the newly made protein – Phosphorylation – Acetylation – Cleavage INSULIN 7. Protein Degradation • If the protein is broken down, obviously it can’t work anymore