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Structure and Replication of DNA John Kyrk Animations • http://www.johnkyrk.com/DNAanatomy.html Are Genes Composed of DNA or Protein? • DNA – Only four nucleotides • thought to have monotonous structure • Protein – 20 different amino acids – greater potential variation – More protein in chromosomes than DNA Bacterial Transformation Experiments Fredrick Griffith (1928) –demonstrate the existence of “Transforming Principle,” a substance able to confer a heritable phenotype from one strain of bacteria to another. Avery MacLeod and McCarty – determine the transforming principle was DNA. Streptococcus Pneumoniae Griffith Experiment Avery Experiment Fig. 16-3 Phage head Tail sheath Tail fiber Bacterial cell 100 nm DNA Hershey Chase Experiment Additional Evidence • Chargaff Ratios • % A = %T and %G = %C (Complexity in DNA Structure) A T G C Arabidopsis 29% 29% 20% 20% Humans 31% 31% 18% 18% Staphlococcus 13% 13% 37% 37% • DNA Content of Diploid and Haploid cells Gametes Humans Chicken 3.25pg 1.267pg Somatic Cells 7.30 pg 2.49pg DNA Friedrich Meischer (1869) extracted a phosphorous rich material from nuclei of which he named nuclein DNA – deoxyribonucleic acid Monomer – Nucleotide Deoxyribose Phosphate Nitrogenous Base (4) Phosphodiester Bond DNA has direction - 5’ and 3’ ends Chromosomes are composed of DNA Fig. 16-UN1 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data Watson and Crick Model • Franklins X-Ray Data – DNA is Double Helix • • • • 2 nm diameter Phosphates on outside 3.4 nm periodicity Bases 0.34nm apart • Watson and Crick – Base Pairing DNA Replication Semiconservative Replication Other Models of Replication Conservative Replication Semi-Conservative Replication Dispersive Replication Culture Bacteria in 15N isotope (DNA fully 15N) 15N DNA One Cell Division in 14N 15N/14N DNA 2nd Cell Division in 14N 14N DNA 15N/14N DNA Less Dense More Dense Density Centrifugation DNA Replication: A Closer Look • The copying of DNA is remarkable in its speed and accuracy • More than a dozen enzymes and other proteins participate in DNA replication Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Video Origins of Replication • At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating • Helicases are enzymes that untwist the double helix at the replication forks • Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template • Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 16-13 Primase Single-strand binding proteins 3 Topoisomerase 5 3 5 Helicase 5 RNA primer 3 DNA Polymerase 5’ 3’ 3’ Pol 5’ Leading and Lagging Strands 3’ 5’ Pol Leading Strand Lagging Strand Pol 3’ RNA Primer 5’ Video 5’ 3’ Other Proteins at Replication Fork 3’ 5’ DNA Pol III Single Stranded Binding Proteins Pol Leading Strand DNA Pol I Ligase Lagging Strand Pol Helicase 3’ 5’ Primase 5’ 3’ Fig. 16-16 Overview Origin of replication Lagging strand Leading strand Lagging strand 2 1 Leading strand Overall directions of replication 3 5 5 Template strand 3 RNA primer 3 5 3 1 5 3 5 Okazaki fragment 3 1 5 3 5 2 3 5 2 3 3 5 1 3 5 1 5 2 1 3 5 Overall direction of replication Damaged DNA Nuclease Excision Repair Nuclease DNA Polymerase Ligase Replicating the Ends of DNA Molecules • Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes • The usual replication machinery provides no way to complete the 5 ends, so repeated rounds of replication produce shorter DNA molecules Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Replicating Ends of Linear Chromosomes Fig. 16-19 5 Ends of parental DNA strands Leading strand Lagging strand 3 Last fragment Previous fragment RNA primer Lagging strand 5 3 Parental strand Removal of primers and replacement with DNA where a 3 end is available 5 3 Second round of replication 5 New leading strand 3 New lagging strand 5 3 Further rounds of replication Shorter and shorter daughter molecules Fig. 16-20 1 µm • If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce • An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Telomerase Chapter 10 From Gene to Protein Protein Synthesis: overview One gene-one enzyme hypothesis (Beadle and Tatum) One gene-one polypeptide (protein) hypothesis Transcription: synthesis of RNA under the direction of DNA (mRNA) Translation: actual synthesis of a polypeptide under the direction of mRNA The “Central Dogma” Flow of genetic information in a cell How do we move information from DNA to proteins? DNA replication RNA protein DNA gets all the glory, but proteins do all the work! trait a a From gene to protein nucleus DNA cytoplasm transcription mRNA a a translation ribosome a a a a a a a a a a a a protein a a a a a a trait Genetic Code Identifying ORF 5’ GACGACGGAUGCGCAAUGCGUCUCUAUGAGACGUAGCUCAC • Locate start codon (1st ATG from 5’ end) • Identify Codons (non overlapping units of three codons including and following start codon) • Stop at stop codon ( remember stop codon doesn’t encode amino acid) • Nucleotides before start codon – 5’UTR • Nucleotides after stop codon 3’UTR • [MetArgAsnAlaSerLeu] The Genetic Code •Use the code by reading from the center to the outside •Example: AUG codes for Methionine Name the Amino Acids • • • • • GGG? UCA? CAU? GCA? AAA? Fig. 17-6 (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene Chromatin Structure Nucleosome (10 nm in diameter) DNA helix in diameter) double (2 nm H1 Histones DNA, the double helix video Histones Histone tail Nucleosomes, or “beads on a string” (10-nm fiber) Fig. 16-21b Chromatid (700 nm) 30-nm fiber Loops Scaffold 300-nm fiber Replicated chromosome (1,400 nm) 30-nm fiber Looped domains (300-nm fiber) Metaphase chromosome 30 nm chromatin fiber 1. Held together by histone tails interacting with neighboring nucleosomes 2. Inhibits transcription 3. Allows DNA replication Gene Expression • • • • Beadle and Tatum Exp Transcription Translation Roles of RNA Beadle and Tatum Isolation of Nutritional Mutants Intermediates in arginine biosynthesis Mutant Ornithine Citrulline Arginine arg-1 + + + arg-2 - + + arg-3 - - + Note: A plus sign means growth; a minus sign means no growth. arg-1 Percursor Ornithine arg-2 arg-3 Citruline Arginine One Gene – One Enzyme One Gene – One Polypeptide The “Central Dogma” Flow of genetic information in a cell How do we move information from DNA to proteins? DNA replication RNA protein DNA gets all the glory, but proteins do all the work! trait Central Dogma of Molecular Biology Protein Synthesis: overview One gene-one enzyme hypothesis (Beadle and Tatum) One gene-one polypeptide (protein) hypothesis Transcription: synthesis of RNA under the direction of DNA (mRNA) Translation: actual synthesis of a polypeptide under the direction of mRNA Transcription from DNA nucleic acid language to RNA nucleic acid language RNA ribose sugar N-bases uracil instead of thymine U:A C:G single stranded lots of RNAs mRNA, tRNA, rRNA, siRNA… DNA transcription RNA Transcription Making mRNA transcribed DNA strand = template strand untranscribed DNA strand = coding strand same sequence as RNA synthesis of complementary RNA strand transcription bubble enzyme RNA polymerase 5 DNA C G 3 build RNA 53 A G T A T C T A rewinding mRNA 5 coding strand G C A G C A T C G T T A 3 G C A U C G U C G T A G C A T A T RNA polymerase C A G C T G A T A T 3 5 unwinding template strand Animation of Transcription • http://vcell.ndsu.nodak.edu/animations/trans cription/movie-flash.htm RNA polymerases 3 RNA polymerase enzymes RNA polymerase 1 only transcribes rRNA genes makes ribosomes RNA polymerase 2 transcribes genes into mRNA RNA polymerase 3 only transcribes tRNA genes each has a specific promoter sequence it recognizes Which gene is read? Promoter region binding site before beginning of gene TATA box binding site binding site for RNA polymerase & transcription factors Enhancer region binding site far upstream of gene turns transcription on HIGH Transcription Factors Initiation complex transcription factors bind to promoter region suite of proteins which bind to DNA hormones? turn on or off transcription trigger the binding of RNA polymerase to DNA Matching bases of DNA & RNA Match RNA bases to DNA bases on one of G the DNA strands G U C A A G C A U G U A C G A U A C 5' RNA A C C polymerase G A U 3' T G G T A C A G C T A G T C A T C G T A C C G T U C Transcription: the process 1.Initiation~ transcription factors mediate the binding of RNA polymerase to an initiation sequence (TATA box) 2.Elongation~ RNA polymerase continues unwinding DNA and adding nucleotides to the 3’ end 3.Termination~ RNA polymerase reaches terminator sequence Eukaryotic genes have junk! Eukaryotic genes are not continuous exons = the real gene expressed / coding DNA introns = the junk inbetween sequence introns come out! intron = noncoding (inbetween) sequence eukaryotic DNA exon = coding (expressed) sequence mRNA splicing Post-transcriptional processing eukaryotic mRNA needs work after transcription primary transcript = pre-mRNA mRNA splicing edit out introns make mature mRNA transcript intron = noncoding (inbetween) sequence ~10,000 base eukaryotic DNA exon = coding (expressed) sequence primary mRNA transcript mature mRNA transcript pre-mRNA ~1,000 base spliced mRNA RNA Processing in Eukaryotes Pre-mRNA (hnRNA) 5’ 3’ Modification of 5’ and 3’ ends 5’CAP Exon1 Intron1 Exon2 Intron2 Exon3 Intron3 Exon4 Spicing of exons Poly A tail 1977 | 1993 Discovery of exons/introns Richard Roberts CSHL Philip Sharp MIT beta-thalassemia adenovirus common cold Splicing must be accurate No room for mistakes! a single base added or lost throws off the reading frame AUGCGGCTATGGGUCCGAUAAGGGCCAU AUGCGGUCCGAUAAGGGCCAU AUG|CGG|UCC|GAU|AAG|GGC|CAU Met|Arg|Ser|Asp|Lys|Gly|His AUGCGGCTATGGGUCCGAUAAGGGCCAU AUGCGGGUCCGAUAAGGGCCAU AUG|CGG|GUC|CGA|UAA|GGG|CCA|U Met|Arg|Val|Arg|STOP| RNA splicing enzymes snRNPs small nuclear RNA proteins exon Spliceosome 5' Whoa! I think we just broke a biological “rule”! snRNPs snRNA intron exon 3' several snRNPs recognize splice site sequence cut & paste gene No, not smurfs! “snurps” spliceosome 5' 3' lariat 5' exon mature mRNA 5' 3' exon 3' excised intron Alternative splicing Alternative mRNAs produced from same gene when is an intron not an intron… different segments treated as exons Starting to get hard to define a gene! More post-transcriptional processing Need to protect mRNA on its trip from nucleus to cytoplasm enzymes in cytoplasm attack mRNA protect the ends of the molecule add 5 GTP cap add poly-A tail longer tail, mRNA lasts longer: produces more protein Translation from nucleic acid language to amino acid language Players in Translation mRNA – Genetic Code Ribosome – synthesizes protien tRNA – adaptor molecule Amino acids Aminoacyl tRNA synthetases - attach amino acids to tRNAs tRNA Transfer RNA structure “Clover leaf” structure anticodon on “clover leaf” end amino acid attached on 3 end Loading tRNA Aminoacyl tRNA synthetase enzyme which bonds amino acid to tRNA bond requires energy ATP AMP bond is unstable so it can release amino acid at ribosome easily Trp activating enzyme C=O OH OH Trp C=O O Trp H 2O tRNATrp anticodon tryptophan attached to tRNATrp O AC C UGG mRNA tRNATrp binds to UGG Ribosomes Facilitate coupling of tRNA anticodon to mRNA codon organelle or enzyme? Structure ribosomal RNA (rRNA) & proteins 2 subunits large small E P A Ribosomes A site (aminoacyl-tRNA site) holds tRNA carrying next amino acid to be added to chain P site (peptidyl-tRNA site) holds tRNA carrying growing polypeptide chain E site (exit site) empty tRNA leaves ribosome from exit site Met U A C A U G 5' E P A 3' Ribosomes How does mRNA code for proteins? TACGCACATTTACGTACGCGG DNA 4 ATCG mRNA AUGCGUGUAAAUGCAUGCGCC 4 AUCG protein ? Met Arg Val Asn Ala Cys Ala 20 How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)? mRNA codes for proteins in triplets DNA TACGCACATTTACGTACGCGG codon mRNA AUGCGUGUAAAUGCAUGCGCC ? protein Met Arg Val Asn Ala Cys Ala Cracking the code 1960 | 1968 Nirenberg & Khorana Crick determined 3-letter (triplet) codon system WHYDIDTHEREDBATEATTHEFATRAT WHYDIDTHEREDBATEATTHEFATRAT Nirenberg (47) & Khorana (17) determined mRNA–amino acid match added fabricated mRNA to test tube of ribosomes, tRNA & amino acids created artificial UUUUU… mRNA found that UUU coded for phenylalanine Marshall Nirenberg 1960 | 1968 Har Khorana The code Code for ALL life! strongest support for a common origin for all life Code is redundant several codons for each amino acid 3rd base “wobble” Why is the wobble good? Start codon AUG methionine Stop codons UGA, UAA, UAG How are the codons matched to amino acids? DNA mRNA 3 TACGCACATTTACGTACGCGG 5 5 3 AUGCGUGUAAAUGCAUGCGCC 3 tRNA amino acid UAC codon 5 Met GCA Arg CAU Val anti-codon Building a polypeptide Initiation brings together mRNA, ribosome subunits, initiator tRNA Elongation adding amino acids based on codon sequence Translocation – Ribosome ratchets over on codon. The tRNA that was in the A site is moved to the P site. The uncharged tRNA in the P site exits the ribosome through the E site. Termination end codon When ribosome reaches the stop codon a release factor binds to the A site and triggers the release of the polypeptide. The ribosome releases the tRNA and the mRNA. 3 2 1 Val Leu Met Met Met Leu Met Leu Ala Leu release factor Ser Trp tRNA UAC 5' C UG A A U mRNA A U G 3' E P A 5' UA C G A C A U G C U GA A U 5' 3' U A C GA C A U G C UG AA U 3' 5' U AC G A C AA U A U G C UG 3' A CC U GG U A A 3' Fig. 17-18-4 Amino end of polypeptide E 3 mRNA Ribosome ready for next aminoacyl tRNA P A site site 5 GTP GDP E E P A P A GDP GTP E P A The Functional and Evolutionary Importance of Introns • Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing • Such variations are called alternative RNA splicing • Because of alternative splicing, the number of different proteins an organism can produce is much greater than its number of genes Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 17-12 Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide Polysomes • Polypeptide synthesis always begins in the cytosol • Synthesis finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ER • Polypeptides destined for the ER or for secretion are marked by a signal peptide • A signal-recognition particle (SRP) binds to the signal peptide • The SRP brings the signal peptide and its ribosome to the ER Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Proteins targeted to ER RNA polymerase DNA Can you tell the story? amino acids exon pre-mRNA intron 5' GTP cap mature mRNA large ribosomal subunit 5' small ribosomal subunit tRNA poly-A tail aminoacyl tRNA synthetase 3' polypeptide tRNA E P A ribosome Prokaryote vs. Eukaryote genes Prokaryotes Eukaryotes DNA in cytoplasm DNA in nucleus circular chromosome linear chromosomes naked DNA DNA wound on histone no introns proteins introns vs. exons introns come out! intron = noncoding (inbetween) sequence eukaryotic DNA exon = coding (expressed) sequence Translation in Prokaryotes Transcription & translation are simultaneous in bacteria DNA is in cytoplasm no mRNA editing ribosomes read mRNA as it is being transcribed Translation: prokaryotes vs. eukaryotes Differences between prokaryotes & eukaryotes time & physical separation between processes takes eukaryote ~1 hour from DNA to protein no RNA processing Mutations Point mutations single base change base-pair substitution silent mutation no amino acid change redundancy in code missense change amino acid nonsense change to stop codon When do mutations affect the next generation? Point mutation leads to Sickle cell anemia What kind of mutation? Missense! Sickle cell anemia Primarily Africans recessive inheritance pattern strikes 1 out of 400 African Americans hydrophilic amino acid hydrophobic amino acid Mutations Frameshift shift in the reading frame changes everything “downstream” insertions adding base(s) deletions losing base(s) Where would this mutation cause the most change: beginning or end of gene? Cystic fibrosis Primarily whites of European descent strikes 1 in 2500 births 1 in 25 whites is a carrier (Aa) normal allele codes for a membrane protein that transports Cl- across cell membrane defective or absent channels limit transport of Cl- (& H2O) across cell membrane thicker & stickier mucus coats around cells mucus build-up in the pancreas, lungs, digestive tract & causes bacterial infections without treatment children die before 5; with treatment can live past their late 20s Deletion leads to Cystic fibrosis delta F508 loss of one amino acid What’s the value of mutations? 2007-2008