2 Metabolism teaches us about genes Metabolic defectsstudying metabolic diseases suggested that genes specified proteinsalkaptonuria (black urine from alkapton)PKU (phenylketonuria)each disease is caused by non-functional enzymeGenes create phenotypeABCDE
4 1 gene – 1 enzyme hypothesis Beadle & TatumCompared mutants of bread mold, Neurospora funguscreated mutations by X-ray treatmentsX-rays break DNAinactivate a genewild type grows on “minimal” mediasugars + required precursor nutrient to synthesize essential amino acidsmutants require added amino acidseach type of mutant lacks a certain enzyme needed to produce a certain amino acidnon-functional enzyme = broken gene
7 Where does that leave us?! So… What is a gene?One gene – one enzymebut not all proteins are enzymesbut all proteins are coded by genesOne gene – one proteinbut many proteins are composed of several polypeptidesbut each polypeptide has its own geneOne gene – one polypeptidebut many genes only code for RNAOne gene – one productbut many genes code for more than one product …Where does that leave us?!
8 if you don’t know what a wabbit looks like. Defining a gene…“Defining a gene is problematic because… one gene can code for several protein products, some genes code only for RNA, two genes can overlap, and there are many other complications.”– Elizabeth Pennisi, Science 2003geneRNAIt’s hard tohunt for wabbits,if you don’t know what a wabbit looks like.1990s -- thought humans had 100,000 genes,000 was considered a good estimate,000,000 is our best estimatepolypeptide 1polypeptide 2polypeptide 3gene
9 let’s go back to genes that code for proteins… The “Central Dogma”How do we move information from DNA to proteins?transcriptiontranslationDNARNAproteinForsimplicity sake,let’s go back to genes that code for proteins…replication
10 From nucleus to cytoplasm… Where are the genes?genes are on chromosomes in nucleusWhere are proteins synthesized?proteins made in cytoplasm by ribosomesHow does the information get from nucleus to cytoplasm?messenger RNAnucleus
11 transcription and translation RNAribose sugarN-basesuracil instead of thymineU : AC : Gsingle strandedmRNA, rRNA, tRNA, siRNA….To get from the chemical language of DNA to the chemical language of proteins requires 2 major stages:transcription and translationtranscriptionDNARNA
12 Transcription Transcribed DNA strand = template strand untranscribed DNA strand = coding strandSynthesis of complementary RNA strandtranscription bubbleEnzymeRNA polymerase
13 Transcription in Prokaryotes InitiationRNA polymerase binds to promoter sequence on DNARole of promoter1. Where to start reading= starting point2. Which strand to read= template strand3. Direction on DNA= always reads DNA 3'5'
14 Transcription in Prokaryotes Promoter sequencesRNA polymerase molecules bound to bacterial DNA
15 Transcription in Prokaryotes ElongationRNA polymerase unwinds DNA ~20 base pairs at a timereads DNA 3’5’builds RNA 5’3’ (the energy governs the synthesis!)No proofreading1 error/105 basesmany copiesshort lifenot worth it!
19 Prokaryote vs. Eukaryote genes ProkaryotesDNA in cytoplasmcircular chromosomenaked DNAno intronsEukaryotesDNA in nucleuslinear chromosomesDNA wound on histone proteinsintrons vs. exonsintron = noncoding (inbetween) sequenceeukaryoticDNAexon = coding (expressed) sequence
20 Transcription in Eukaryotes 3 RNA polymerase enzymesRNA polymerase Ionly transcribes rRNA genesRNA polymerase I Itranscribes genes into mRNARNA polymerase I I Ieach has a specific promoter sequence it recognizes
21 Transcription in Eukaryotes Initiation complextranscription factors bind to promoter region upstream of geneproteins which bind to DNA & turn on or off transcriptionTATA box binding siteonly then does RNA polymerase bind to DNA
22 Post-transcriptional processing Primary transcripteukaryotic mRNA needs work after transcriptionProtect mRNAfrom RNase enzymes in cytoplasmadd 5' capadd polyA tailEdit out intronsA3' poly-A tailCH3mRNA5'5' cap3'GPA’sintron = noncoding (inbetween) sequenceeukaryoticDNAexon = coding (expressed) sequencepre-mRNAprimary mRNAtranscriptmature mRNAtranscriptspliced mRNA
23 Transcription to translation Differences between prokaryotes & eukaryotestime & physical separation between processesRNA processing
24 Translation in Prokaryotes Transcription & translation are simultaneous in bacteriaDNA is in cytoplasmno mRNA editing needed
25 DNA mRNA protein From gene to protein transcription translation aatranscriptiontranslationDNAmRNAproteinribosomemRNA leaves nucleus through nuclear poresproteins synthesized by ribosomes using instructions on mRNAnucleuscytoplasm
26 How does mRNA code for proteins? TACGCACATTTACGTACGCGGDNAAUGCGUGUAAAUGCAUGCGCCmRNA?Met Arg Val Asn Ala Cys AlaproteinHow can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)?
27 Cracking the code 1960 | 1968 Nirenberg & Matthaei determined 1st codon–amino acid matchUUU coded for phenylalaninecreated artificial poly(U) mRNAadded mRNA to test tube of ribosomes, tRNA & amino acidsmRNA synthesized single amino acid polypeptide chainphe–phe–phe–phe–phe–phe
29 TranslationCodonsblocks of 3 nucleotides decoded into the sequence of amino acids
30 mRNA codes for proteins in triplets TACGCACATTTACGTACGCGGDNAAUGCGUGUAAAUGCAUGCGCCmRNAAUGCGUGUAAAUGCAUGCGCCmRNA?Met Arg Val Asn Ala Cys Alaprotein
31 The code For ALL life! Code is redundant Why is this a good thing? strongest support for a common origin for all lifeCode is redundantseveral codons for each amino acidWhy is this a good thing?Strong evidence for a single origin in evolutionary theory.Start codonAUGmethionineStop codonsUGA, UAA, UAG
32 How are the codons matched to amino acids? 3'5'TACGCACATTTACGTACGCGGDNA5'AUGCGUGUAAAUGCAUGCGCC3'mRNAcodon3'5'UACMettRNAGCAArgamino acidCAUValanti-codon
33 cytoplasm transcription translation protein nucleus
34 tRNA structure “Clover leaf” structure anticodon on “clover leaf” end amino acid attached on 3' end
35 Loading tRNA Aminoacyl tRNA synthetase enzyme which bonds amino acid to tRNAendergonic reactionATP AMPenergy stored in tRNA-amino acid bondunstableso it can release amino acid at ribosomeThe 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.
36 Ribosomes Facilitate coupling of tRNA anticodon to mRNA codon organelle or enzyme?Structureribosomal RNA (rRNA) & proteins2 subunitslargesmall
37 Ribosomes P site (peptidyl-tRNA site) A site (aminoacyl-tRNA site) holds tRNA carrying growing polypeptide chainA site (aminoacyl-tRNA site)holds tRNA carrying next amino acid to be added to chainE site (exit site)empty tRNA leaves ribosome from exit site
38 Building a polypeptide Initiationbrings together mRNA, ribosome subunits, proteins & initiator tRNAElongationTermination
46 Universal code Code is redundant several codons for each amino acid “wobble” in the tRNA“wobble” in the aminoacyl-tRNA synthetase enzyme that loads the tRNAStrong evidence for a single origin in evolutionary theory.
47 Mutations Point mutations single base change base-pair substitution silent mutationno amino acid changeredundancy in codemissensechange amino acidnonsensechange to stop codonWhen do mutations affect the next generation?
48 Point mutation leads to Sickle cell anemia What kind of mutation?
53 Transcription -- another look The process of transcription includes many points of controlwhen to start reading DNAwhere to start reading DNAwhere to stop reading DNAediting the mRNAprotecting mRNA as it travels through cell
54 Primary transcript Processing mRNA protecting RNA from RNase in cytoplasmadd 5’ capadd polyA tailremove intronsAUGUGA
55 Protecting RNA 5’ cap added 3’ poly-A tail added G trinucleoside (G-P-P-P)protects mRNAfrom RNase (hydrolytic enzymes)3’ poly-A tail addedA’shelps export of RNA from nucleusUTRUTR
56 Dicing & splicing mRNA Pre-mRNA mRNA edit out introns intervening sequencessplice together exonsexpressed sequencesIn higher eukaryotes90% or more of gene can be intronno one knows why…yetthere’s a Nobel prize waiting…“AVERAGE”…“gene” = 8000bpre-mRNA = 8000bmature mRNA = 1200bprotein = 400aalotsa “JUNK”!average size gene (transcription unit) = basesaverage size primary transcript = 8000 basesaverage size mature RNA = 1200 basesaverage size protein = 400 amino acidslots of “junk DNA”
57 Discovery of Split genes 1977 | 1993Discovery of Split genesRichard RobertsPhilip SharpadenovirusNE BioLabsMITcommon cold
58 Splicing enzymes snRNPs Spliceosome RNA as ribozyme several snRNPs small nuclear RNARNA + proteinsSpliceosomeseveral snRNPsrecognize splice site sequencecut & pasteRNA as ribozymesome mRNA can splice itselfRNA as enzyme
59 Ribozyme 1982 | 1989 RNA as enzyme Sidney Altman Thomas Cech Yale U of Colorado
60 Splicing details No room for mistakes! editing & splicing must be exactly accuratea 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|
61 Alternative splicing Alternative mRNAs produced from same gene when is an intron not an intron…different segments treated as exonsHardto define a gene!
62 Domains Modular architecture of many proteins separate functional & structural regionscoded by different exons in same “gene”
63 The Transcriptional unit (gene?) enhancer1000+btranslationstarttranslationstopexons20-30btranscriptional unitRNApolymerase3'TACACT5'TATADNAtranscriptionstartUTRintronstranscriptionstopUTRpromoterDNApre-mRNA5'3'mature mRNA5'3'GTPAAAAAAAA
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