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55-213 Lecture 21 notes.pdf
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Department
Biological Sciences
Course
55-213
Professor
Hubberstey
Semester
Winter

Description
Lecture 21 2013-01-10 2:29 PM Chapter)20:)Eukaryotic)Transcription' • This'is'probably'one'of'the'more'complicated'lectures.'Transcription'in'eukaryotes' needs'many'more'sequences'than'in'bacteria.'' • In'bacteria,'there'are'RNA'polymerases'that'have'different'sigma'factors'(aka' promoters)'as'well'as'proteins'called'suppressors'that'can'block'access'of'the'RNA' polymerase'to'the'promoters' • In'eukaryotes,'promoters'are'defined'by'many'more'sequences'than'in'bacteria' o In'eukaryotes,'it’s'a'lot'more'complicated.'We'have'a'lot'more'genes,'we' have'compartmentalization,'we'have'different'types'of'genes'and'RNA' polymerases.'There'are'thousands'of'protein'factors'encoded'by'different' genes,'which'are'responsible'for'these'processes,'both'positive'and'negative' (i.e.'turning'a'gene'on'or'off)' o Wtranscription'other'than'RNA'polymerase.'Hence,'we’ve'come'so'far'in'such'a' short'period'of'time'in'terms'of'our'understanding'of'how'transcription'is' turned'on'in'our'cells' o Our'promoters'are'much'more'complex'than'in'bacteria.'We'also'have'a'lot' of'different'types'of'sequences'that'bind'different'promoters'than'in'bacteria' • Sequences'that'bind'transcription'factors'are'throughout'promoter'and'can'exist'>' 1000'bps'away'from'initiation'site' o Unlike'bacteria'where'all'of'the'proteins'are'relatively'close'to'the' transcription'start'site,'in'eukaryotes,'there'are'binding'sites'for'transcription' factors'that'are'thousands'of'bp'away' o This'presents'a'problem'to'our'understanding'of'how'transcription'occurs,' because'sequences'can'be'anywhere'(i.e.'even'thousands'of'bases' upstream).'There'are'also'binding'sites'within'the'gene'that'regulate'when' that'particular'gene'is'active.'' • Transcription'factors'required'for'initiation'but'not'elongation' o The'other'important'point'to'make'is'that'transcription'factors'are'primarily' just'required'to'turn'the'gene'on.'They’re'not'required'for'the'elongation' part.'Thus,'when'RNA'polymerase'has'started'and'elongation'starts,'the' transcription'factors'come'off' • RNA'polymerase'binds'to'startpoint'in'promoter'with'help'from'other'proteins' o How'does'the'RNA'polymerase'know'where'a'promoter'is?'There'are'3' billion'bp'of'DNA'in'our'genome'–'only'a'very'small'amount'of'these'encode' promoters'or'sequences'that'drive'expression'of'genes.'Thus,'how'does'it' know'where'to'bind?'Even'more'complicated'are'the'transcription'factors' that'have'to'communicate'with'the'RNA'polymerase'molecule,'which'is' always'have'near'the'transcription'start'site.'How'does'this'happen?'How'do' some'genes'have'binding'sites'for'up'to'100'different'proteins?'How'is'all' this'regulated?' • See'figure'20.1'!'Transcription'is'controlled'by'a'promoter'and'an'enhancer' o The'left'is'the'start'site,'where'RNA'polymerase'initiates'transcription' o About'200'bp'upstream'is'a'whole'bunch'of'sequences'that'define'where'the' promoter'is' o The'complicated'factor'is'that'we'have'enhancers'on'the'left,'that'bind' specific'transcription'factors'that'can'enhance'the'level'of'initiation'and'also' drive'the'expression'of'a'lot'of'genes'several'fold.'If'enhancers'are'deleted'or' mutated,'transcription'goes'way'down.'There'are'signals'given'from'the' enhancer'that'tell'the'RNA'polymerase'and'the'promoter'to'start' transcription' o The'red'circles'are'proteins'that'bind'to'specific'DNA'sequences'–'that’s'how' we'know'there’s'a'promoter'there' ' ' ' ' ' ' ' ' Eukaryotic)RNA)Polymerases:' • The'three'types'of'RNA'polymerase'encode'the'3'different'types'of'RNA'–'this'is' different'from'bacteria'which'have'only'1'type' • 3'RNA'polymerases'in'eukaryotes' o Pol'I:'rRNA' o Pol'II:'mRNA' o Pol'III:'tRNA'and'snRNA' • RNA'polymerases'have'>10'subunits' o Some'unique,'some'common' o Very'large'protein'complex'that'requires'all'the'proteins'in'it'to'come' together'to'form'the'active'RNA'polymerase' • See'figure'20.2' o If'you'take'the'RNA'polymerases'and'run'them'on'a'gel,'you'see'something' like'this' o Here'you'have'some'of'the'larger'subunits'in'the'100c200'kDA'range' o The'smaller'ones'are'more'common'(~'25'kDa)'region' ' ' ' ' ' ' ' ' ' ' ' ' • Largest'polymerase'subunit'of'pol'II'has'carboxycterminal'domain'(CTD)'that'has' several'amino'acid'repeat'sequences'(YSPTSPS)'='tyrosine,'serine,'proline,' threonine,'serine,'proline,'serine' o The'one'at'the'top'of'the'gel' o Is'a'very'important'subunit'of'RNA'pol'II' o What'is'unique'about'that'amino'acid'sequence?'It'is'all'alpha'amino'acids,' which'are'able'to'divide'postctranslation'!'they'can'be'modified'by' phosphorylation,'which'can'occur'on'tyrosine,'serine,'threonine' o Hence,'it’s'a'heavily'phosphorylated'sequence'called'CTD'that'is'repeated' over'and'over'again'' • Yeast'has'26'repeats,'mammals'~50' • Mutations'that'remove'repeats'are'lethal'in'yeast' o If'you'take'the'sequences'off'and'produce'a'large'subunit'without'CTD,'the' cell'will'die,'as'seen'in'yeast.'Hence,'that'sequence'is'extremely'important!' • CTD'region'is'highly'phosphorylated'on'Ser'and'Thr'residues'during'initiation' o You'can’t'get'initiation'unless'you'get'phosphrylation'of'the'sequence' o This'is'one'of'the'final'triggers'that'signal'to'RNA'polymerase'that' transcription'is'ready'to'go' o Hence,'if'you'block'phosphorylation,'you'block'initiation'and'thus' transcription'' • All'eukaryotic'pol'II'enzymes'are'inhibited'by'acamanitin'(from'mushroom'Amanita)' o Other'organisms'have'evolved'to'block'these'enzymes.'If'you'eat'that' mushroom,'you’re'dead'because'it'will'block'phosphorylation'and'hence'pol' II'enzymes'needed'for'transcription' • Next'we'will'look'at'the'different'promoters'of'RNA'pol'I,'pol'II'and'pol'III.'The' promoters'are'different'and'set'up'differently.'This'makes'sense.'' • We'don’t'have'sigma'factors'like'bacteria'do'–'sigma'factors'are'used'in'bacteria'to' distinguish'between'different'promoters.'In'eukaryotes,'there'is'a'different' mechanism'by'making'different'RNA'polymerases'that'recognize'different' promoters.'Hence,'there’s'a'higher'level'of'regulation'in'eukaryotes'than' prokaryotes' ' RNA)polymerase)I)promoters:' • RNA'pol'I'exists'as'holoenzyme'in'complex'with'other'factors'to'initiate'transcription' of'rRNA'genes' • Some'subunits'are'unique'to'pol'I,'others'are'common'to'the'others.'Its'required'to' initiate'transcription'for'ONLY'ribosomal'RNA'genes' • Pol'I'promoters'contain'“core'promoter”'that'is'GC'rich'and'is'enough'for'initiation' o Found'in'ribosomal'RNA'genes' • Core'promoter'also'has'AT'rich'element'called'Inr'at'startpoint' o It'also'has'a'particular'AT'rich'element'called'Inr'which'is'found'right'at'the' startsite'where'the'first'nucleotide'is'put'into'the'ribosomal'RNA'transcript.' Remember,'reibosomal'RNA'is'NOT'translated!'Its'single'stranded'RNA'that' gets'incorporated'into'the'final'structure'of'the'ribosome'(which'is'a'series'of' different'proteins'that'bind'with'the'rRNA'to'form'a'complex'called'the' ribosome)' • Second'“upstream'promoter'element”'(UPE)'lies'~50bp'upstream'from'core' promoter' o So'its'at'c50'from'the'startsite' o It'was'relatively'easy'to'pull'out'these'genes,'since'there'are'only'a'few' ribosomal'RNA'genes'but'they’re'transcribed'at'an'exceptionally'high'level.' This'accounts'for'the'vast'majority'of'all'the'RNA'in'our'cells.'If'you'isolate'all' of'our'RNA,'about'80c90%'of'it'would'be'ribosomal'RNA.'This'is'because' they’re'used'for'everything:'especially'in'translation'to'make'proteins' o Molecular'biologists'wanted'to'know:'are'there'unique'sequences'or' proteins'that'can'affect'the'initiation'of'transcription?'There'are'!'UBF' • Specific'factor'(UBF)'binds'to'UPE'and'brings'core'promoter'closer'to'UPE' o This'is'a'particular'protein'that'binds'to'the'UPE'element'and'takes'the'DNA' out'so'that'all'the'factors'are'now'next'to'each'other'(interacting'with'RNA' pol'at'the'initiatation'site).'When'all'of'this'is'set'up'properly,'initiation'can' begin' • SL1'factor'then'binds'to'core'promoter'and'makes'RNA'pol'I'bind'at'startpoint' o Positioning'factors'are'proteins'that'tell'the'RNA'polymerase'“here’s'a' promoter,'and'if'you'bind'to'me,'you’re'at'the'right'spot”' o All'3'promoters'and'RNA'pol’s'have'different'positioning'factors,'which'are' very'important'' o In'RNA'pol'I,'the'positioning'factor'is'called'SL1' • See'figure'20.5'!'Pol'I'promoters'have'2'sequence'components' o Startpoint'is'on'the'right' o Core'promoter'that'is'GC'rich'and'AT'rich'is'nearby' o There'are'also'additional'promoter'factors'shown'in'purple'(the'UPE)'that' bind'this'UBF' o The'purple'part'loops'out'the'DNA,'and'other'proteins'like'SL1'come'in'and' bind'at'the'core'promoter'and'position'RNA'polymerase'(shown'in'red)'at' this'correct'site' o At'the'end,'all'these'specific'sequences'come'together.'When'they’re'there' in'the'right'proportions,'it'sends'a'signal'to'RNA'polymerase'that'now'is'the' time'to'start'transcribing'' ' ' ' ' ' ' ' ' ' ' ' ' ' ' RNA)polymerase)II)promoters:' • It'gets'kind'of'stupid'because'there'are'so'many'factors'involved.'The'vast'majority' of'our'genes'in'cells'are'transcribed'by'RNA'pol'II.'But,'DNA'looks'the'same'(genome' is'exactly'the'same)'in'our'brain,'liver,'kidney,'etc'(i.e.'in'any'tissue'of'our'body).'So' why'are'certain'genes'expressed'only'in'the'brain,'or'liver,'etc?'They’re'only' transcribed'in'those'tissues'because'maybe,'there'are'transcription'factors'found'in' those'tissues' • There'are'hundreds'of'transcription'factors'involved'in'regulating'when'these' different'genes'get'turned'on'in'the'different'tissues,'and'more'importantly,'during' development' • If'screwed'up,'you’re'done' • Today,'we'will'discuss'how'or'what'are'the'factors'that'allow'RNA'pol'II'to'recognize' a'particular'promoter?''''' • Transcription'from'pol'II'promoters'requires'pol'II'and'“basal'factors”'!'TF X.'X' identifies'factor'as'a'letter'(AcJ).' II o It'requires'2'sets'of'factors:'the'fundamental'proteins'known'as'“basal' factors”,'and'the'specific'factors'' o The'nomenclature'is'TF'for'transcription'factor,'a'subscript'for'the'associated' RNA'polymerase,'and'X'representing'a'letter'(AcJ)'of'a'basal'factor'involved' in'transcription'' o The'problem'is'we'still'didn’t'understand'how'this'worked'–'it’s'not'like'A' came'first,'followed'by'B'and'ending'with'J.'So'don’t'think'about'it'as'an' alphabet.'Rather,'they'all'have'to'be'there'for'initiation'to'start' • Core'promoter'consists'of'2'elements:' o Inr:'initiator'region'–'Py CAP2 '(seq5ences'c3'to'+5)' " Py'stands'for'pyrimidine'' " This'is'a'marker,'ie'a'flag,'that'this'is'an'initiation'region' o TATABOX:'~'c25'(similar'to'c10'region'in'bacteria)' " In'eukaryotes,'the'TATABOX'is'a'bit'further'back'than'the'startsite'' o DPE:'downstream'promoter'elementcfound'in'TATAcless'promoters' (sequence'+28'to'+32)' " This'is'a'downstream'promoter'element,'meaning'to'the'right'of'the' startsite'(a'positive'number).'Its'not'found'in'all'promoters'–'only'in' those'that'DON’T'have'tataboxes.'This'was'found'when'people' realized'that'you'can'still'get'transcription'without'a'tatabox' " Upstream'='left'of'the'startsite'='negative'number' o Pol'II'is'similar'to'other'promoters,'except'there’s'a'Inr'region' o All'of'these'sequences'are'important'because'they'bind'particular'factors' that'allow'RNA'polymerase'to'recognize'where'the'promoter'is.'Its'all'based' on'proteincDNA'interactions'and'proteincprotein'interactions'of'all'the' various'transcription'factors' • Promoters'can'be:'''''Inr'+'TATA'''''or'''''''Inr'+'DPE' o You'have'to'have'an'initiatior'and'a'tatabox,'or'an'initiator'and'a'DPE' o You'can'only'have'2,'not'all'three' o All'have'to'contain'an'initiator'' • See'figure'20.7'!'a'minimal'pol'II'promoter'has'only'2'elements' o Shows'you'what'a'basic'pol'II'promoter'looks'like' o The'middle'region'is'the'startoint,'the'Inr'region'with'the'7'pyrimidines'and' CA' o Upstream'theres'a'TATAbox,'and'downstream'there’s'a'DPE' o These'are'the'only'types'of'promoters'you'can'have;'either'the'one'on'the' left'or'the'one'on'the'right'' ' ' ' ' ' ' ' ' Pol)II)Factors:' • All'polymerases'require'positioning'factor:' o SL1'for'pol'I' o TF B'III'pol'III' • For'pol'II'it'is'TIID:' o TATA'binding'factor'(TBP)'+'11'TAFs'(TBPcassociated'factors)' " TF IIis'a'positioning'factor' " TF IIis'a'large'component'of'different'proteins'–'a'TATA'binding' factor'(TBP)'and'11'factors'associated'to'the'former'' " TBP'is'the'key'positioning'factor'for'pol'II' o TAF 00IIhere'00'represents'molecular'weight'of'factor'(e.g.'TAF 230)' II " TAFs'are'written'as'TAF 00,IIhere'the'number'represents'the' molecular'weight'(instead'
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