2 What do genes code for? How does DNA code for cells & bodies? DNA how are cells and bodies made from the instructions in DNADNAproteinscellsbodies
3 transcription and translation The “Central Dogma”Flow of genetic information in a cellHow do we move information from DNA to proteins?transcriptiontranslationDNARNAproteintraitTo get from the chemical language of DNA to the chemical language of proteins requires 2 major stages:transcription and translationreplication
4 one gene : one enzyme hypothesis 1941 | 1958Beadle & Tatumone gene : one enzyme hypothesisGeorge BeadleEdward Tatum"for their discovery that genes act by regulating definite chemical events"
5 Beadle & Tatum Wild-type Neurospora Minimal medium Select one of the sporesGrow oncomplete mediumcontrolNucleicacidCholinePyridoxineRiboflavinArginineMinimal media supplemented only with…ThiamineFolicNiacinInositolp-Aminobenzoic acidTest on minimalmedium to confirmpresence of mutationGrowth oncompleteX rays or ultraviolet lightasexualsporescreate mutationspositive controlnegative controlmutation identifiedexperimentalsamino acid supplements
7 Metabolism taught us about genes Inheritance of metabolic diseasessuggested that genes coded for enzymeseach disease (phenotype) is caused by non-functional gene productmetabolic pathwaydiseasediseasediseasediseaseABCDEenzyme 1enzyme 2enzyme 3enzyme 4
8 from DNA nucleic acid language to RNA nucleic acid language Transcriptionfrom DNA nucleic acid language to RNA nucleic acid language
9 DNA RNA RNA ribose sugar N-bases single stranded lots of RNAs uracil instead of thymineU : AC : Gsingle strandedlots of RNAsmRNA, tRNA, rRNA, snRNA…transcriptionDNARNA
10 Transcription Making mRNA transcribed DNA strand = template strand untranscribed DNA strand = coding strandsame sequence as RNAsynthesis of complementary RNA strandtranscription bubbleenzymeRNA polymerasecoding strand3AGCATCGT5AGAAAGTCTTCTCATACGDNAT3CGTAAT5GGCAUCGUT3CunwindingGTAGCArewindingmRNARNA polymerasetemplate strandbuild RNA 535
11 Problem?????Only 4 nucleotide bases to make 20 amino acids?How?
12 WHYDIDTHEREDBATEATTHEFATRAT WHYDIDTHEREDBATEATTHEFATRAT Cracking the codeMarshall Nirenberg (1961)determined 3-letter (triplet) codon systemWHYDIDTHEREDBATEATTHEFATRATWHYDIDTHEREDBATEATTHEFATRATdetermined mRNA–amino acid matchadded fabricated mRNA to test tube of ribosomes, tRNA & amino acidscreated artificial UUUUU… mRNAfound that UUU coded for phenylalanine
14 The code Code for ALL life! Code is redundant Start codon Stop codons strongest support for a common origin for all lifeCode is redundantseveral codons for each amino acid3rd base “wobble”Strong evidence for a single origin in evolutionary theory.Start codonAUGmethionineStop codonsUGA, UAA, UAG
15 DNA: TAC CTT GTG CAT GGG ATC mRNA AUG GAA CAC GUA CCC UAG A.A MET G.A HIS VAL PRO STOP15
19 Which gene is read? Promoter region Enhancer region binding site before beginning of geneTATA box binding sitebinding site for RNA polymerase & transcription factorsEnhancer regionbinding site far upstream of geneturns transcription on HIGH
20 Transcription Factors Initiation complextranscription factors bind to promoter regionsuite of proteins which bind to DNAhormones?turn on or off transcriptiontrigger the binding of RNA polymerase to DNA
21 Matching bases of DNA & RNA Match RNA bases to DNA bases on one of the DNA strandsCUGAGUGUCUGCAACUAAGCRNApolymeraseU5'A3'GACCTGGTACAGCTAGTCATCGTACCGT
24 5’ = gets guanine cap3’ = gets adenine cap* Exit the envelope, direct ribosome.
25 Eukaryotic genes have junk! Eukaryotic genes are not continuousexons = the real geneexpressed / coding DNAintrons = the junkinbetween sequenceintron = noncoding (inbetween) sequenceeukaryotic DNAexon = coding (expressed) sequence
26 mRNA splicing Post-transcriptional processing eukaryotic mRNA needs work after transcriptionprimary transcript = pre-mRNAmRNA splicingedit out intronsmake mature mRNA transcripteukaryotic RNA is about 10% of eukaryotic gene.intron = noncoding (inbetween) sequence~10,000 baseseukaryotic DNAexon = coding (expressed) sequencepre-mRNAprimary mRNAtranscript~1,000 basesmature mRNAtranscriptspliced mRNA
27 How is pre-mRNA spliced? Specific sequence at ends of introns signal ‘small nuclear ribonucleoproteins’ called snRNP’s (snurps). Spliceosome
29 Discovery of exons/introns 1977 | 1993Discovery of exons/intronsRichard RobertsPhilip SharpBeta thalassemia is an inherited blood disorder that reduces the production of hemoglobin. Symptoms of beta thalassemia occur when not enough oxygen gets to various parts of the body due to low levels of hemoglobin and a shortage of red blood cells (anemia).Signs and symptoms of thalassemia major appear in the first 2 years of life. Infants have life-threatening anemia and become pale and listless. They also have a poor appetite, grow slowly, and may develop yellowing of the skin and whites of the eyes (jaundice). The spleen, liver, and heart may be enlarged, and bones may be deformed. Adolescents with thalassemia major may experience delayed puberty.Thalassemia is a quantitative problem of too few globins synthesized, whereas sickle-cell anemia is a qualitative problem of synthesis of an incorrectly functioning globin.adenovirusCSHLMITcommon coldbeta-thalassemia
30 Splicing must be accurate No room for mistakes!a single base added or lost throws off the reading frameAUGCGGCTATGGGUCCGAUAAGGGCCAUAUGCGGUCCGAUAAGGGCCAUAUG|CGG|UCC|GAU|AAG|GGC|CAUMet|Arg|Ser|Asp|Lys|Gly|HisAUGCGGCTATGGGUCCGAUAAGGGCCAUAUGCGGGUCCGAUAAGGGCCAUAUG|CGG|GUC|CGA|UAA|GGG|CCA|UMet|Arg|Val|Arg|STOP|
31 we just broke a biological “rule”! Whoa! I thinkwe just broke a biological “rule”!RNA splicing enzymessnRNPssmall nuclear RNAproteinsSpliceosomeseveral snRNPsrecognize splice site sequencecut & paste genesnRNPsexonintronsnRNA5'3'spliceosomeexonexcisedintron5'3'lariatmature mRNANo, not smurfs!“snurps”
32 Starting to get hard to define a gene! Alternative splicingAlternative mRNAs produced from same genewhen is an intron not an intron…different segments treated as exonsStarting to get hard to define a gene!
33 More post-transcriptional processing Need to protect mRNA on its trip from nucleus to cytoplasmenzymes in cytoplasm attack mRNAprotect the ends of the moleculeadd 5 GTP capadd poly-A taillonger tail, mRNA lasts longer: produces more proteineukaryotic RNA is about 10% of eukaryotic gene.A3' poly-A tailmRNA5'5' cap3'GPA’s
34 DNA mRNA protein trait From gene to protein nucleus cytoplasm aaFrom gene to proteinnucleuscytoplasmtranscriptiontranslationDNAmRNAproteinribosometrait
35 from nucleic acid language to amino acid language Translationfrom nucleic acid language to amino acid language
36 How does mRNA code for proteins? TACGCACATTTACGTACGCGGDNA4ATCGAUGCGUGUAAAUGCAUGCGCCmRNA4AUCG?Met Arg Val Asn Ala Cys Alaprotein20How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)?
37 mRNA codes for proteins in triplets TACGCACATTTACGTACGCGGDNAcodonAUGCGUGUAAAUGCAUGCGCCmRNAAUGCGUGUAAAUGCAUGCGCCmRNA?Met Arg Val Asn Ala Cys Alaprotein
38 How are the codons matched to amino acids? 35DNATACGCACATTTACGTACGCGG53mRNAAUGCGUGUAAAUGCAUGCGCCcodon35UACMetGCAArgtRNACAUValanti-codonamino acid
39 DNA mRNA protein trait From gene to protein nucleus cytoplasm aaFrom gene to proteinnucleuscytoplasmtranscriptiontranslationDNAmRNAproteinribosometrait
40 Transfer RNA structure “Clover leaf” structureanticodon on “clover leaf” endamino acid attached on 3 end
41 tryptophan attached to tRNATrp tRNATrp binds to UGG condon of mRNA Loading tRNAAminoacyl tRNA synthetaseenzyme which bonds amino acid to tRNAbond requires energyATP AMPbond is unstableso it can release amino acid at ribosome easilyThe tRNA-amino acid bond is unstable. This makes it easy for the tRNA to later give up the amino acid to a growing polypeptide chain in a ribosome.TrpC=OTrpTrpC=OOHH2OOHOC=OOactivatingenzymetRNATrpACCUGGmRNAanticodontryptophan attached to tRNATrptRNATrp binds to UGG condon of mRNA
42 Ribosomes Facilitate coupling of tRNA anticodon to mRNA codon organelle or enzyme?Structureribosomal RNA (rRNA) & proteins2 subunitslargesmallEPA
43 Ribosomes A site (aminoacyl-tRNA site) P site (peptidyl-tRNA site) holds tRNA carrying next amino acid to be added to chainP site (peptidyl-tRNA site)holds tRNA carrying growing polypeptide chainE site (exit site)empty tRNA leaves ribosome from exit siteMetUAC5'UGA3'EPA
44 Building a polypeptide 123Building a polypeptideInitiationbrings together mRNA, ribosome subunits, initiator tRNAElongationadding amino acids based on codon sequenceTerminationend codonLeuValreleasefactorSerMetMetMetMetLeuLeuLeuAlaTrptRNACAGUACUACGACACGACA5'U5'UACGAC5'AAAUGCUGUAUGCUGAUAUGCUGAAU5'AAUmRNAAUGCUG3'3'3'3'ACCUGGUAAEPA3'
45 start of a secretory pathway Destinations:secretionnucleusmitochondriachloroplastscell membranecytoplasmetc…Protein targetingSignal peptideaddress labelstart of a secretory pathway
46 Can you tell the story? RNA polymerase DNA amino acids tRNA pre-mRNA exonintrontRNApre-mRNA5' GTP capmature mRNAaminoacyl tRNA synthetasepoly-A tail3'large ribosomal subunitpolypeptide5'tRNAsmall ribosomal subunitEPAribosome
47 The Transcriptional unit (gene?) enhancer1000+btranslationstarttranslationstopexons20-30btranscriptional unit (gene)RNApolymerase3'TACACT5'TATADNAtranscriptionstartUTRintronstranscriptionstopUTRpromoterDNApre-mRNA5'3'mature mRNA5'3'GTPAAAAAAAA
48 Protein Synthesis in Prokaryotes Bacterial chromosomeProtein Synthesis in ProkaryotesTranscriptionmRNAPsssst… no nucleus!CellmembraneCell wall
49 Prokaryote vs. Eukaryote genes ProkaryotesDNA in cytoplasmcircular chromosomenaked DNAno intronsEukaryotesDNA in nucleuslinear chromosomesDNA wound on histone proteinsintrons vs. exonsWalter Gilbert hypothesis:Maybe exons are functional units and introns make it easier for them to recombine, so as to produce new proteins with new properties through new combinations of domains.Introns give a large area for cutting genes and joining together the pieces without damaging the coding region of the gene…. patching genes together does not have to be so precise.introns come out!intron = noncoding (inbetween) sequenceeukaryoticDNAexon = coding (expressed) sequence
50 Translation in Prokaryotes Transcription & translation are simultaneous in bacteriaDNA is in cytoplasmno mRNA editingribosomes read mRNA as it is being transcribed
51 Translation: prokaryotes vs. eukaryotes Differences between prokaryotes & eukaryotestime & physical separation between processestakes eukaryote ~1 hour from DNA to proteinno RNA processing