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BIOL300 FALL 2013(Midterm notes until lec 10).pdf

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McGill University
Anatomy & Cell Biology
ANAT 262
Frieder Schoeck

BIOL300 FALL 2013 Lecture 2Readings Chap 13 pg 429447 Chap 19 page 615632 Chap 20 p707708 Transcription can be divided into 3 main phasesINITIATION o RNA pol binds to the duplex DNA at a specific site called the promoterClosed complexcomplex RNA pol and DNA o Pol denatures the DNA locally near the start site of transcription forming a transcription bubble Closed complex is converted to OPEN COMPLEX where the DNA is singlestranded o POl then catalyzes formation of phospodiester bondlinkage between the first two rNTPs ELONGATION o Pol moves in 35 direction along the template strand melting the duplex DNA adding rNTPS sequentially togrowing RNATERMINATION o RNA pol recognizes a top site and at that point it will release both the DNA and the newly synthesized RNATranscription can be regulated at all 3 phases of transcription but regulation of initiation is by far the most common ReadingsProkaryotic RNA polymerase has various subunitsMade up of several polypeptidesEach of the subunits has a specific functionConsists of 2 alpha and 2 beta subunits and 1 sigma subunit o Alpha subunits initiate assembly of complex and bind regulatory proteins eg cAMPCAP stimulates transcription and can interact with the alpha subunits o Beta subunits polymerize NTPs and involved in catalyzation offormation of phosphodiester bonds between the rNTPs o Sigma subunit important in recognizing the promoter hence determining where transcription can start on DNA moleculeDoesnt stay attached to the RNA pol once elongation phase is underway After about 10 base pairs have been transcribedthe sigma subunit dissociates from the core polymerase complex o SPACE MODEL FOR TRANSCRIBING RNA POLRNADNA complex sits between the beta and betaprim subunitsSigma factors of EColi recognize different sequencesSigma 70 recognizes on duplex DNA the TATA box located 10 nts upstream of the transcriptional start site and thesequence TTGACA located at 35 Differences in Ecoli promoter sequences affect the frequency of transcription initiationNot all the promoter sequences are the same Those sequences only constitute a consensus element Promoter that diverges from the consensus sequence will be weaker This allows for regulation by other proteins that can affect the ability of sigma 70 to recognize DNAse I footprinting is a technique reveals control element sequencesDNAse I is an endonuclease that cleaves DNADNA which is bound by a regulatory protein cannot be cleaved by DNase IFragments of DNA known to contain a regulatory sequence are labelled with radioactive 32Pradiolabelled nt at 5end onlySamples of DNA fragments are digested in the presence and absence of protein sample which bind to the unknown regulatory sequenceA low concentration of DNAse I is used so that on average each DNA molecule is cleaved only once o DNAse I randomly cleaves the DNA moleculeIf the protein sample does not contain the cognate protein the DNA fragment is cleaved at multiple positions between the labelled and unlabelled endSample AIf the protein sample contains a cognate protein the protein binds to the DNA protecting the fragment from digestion by DNAse I Following DNAse I treatment the DNA fragments are isolatedFragments are run ona gel to separate them by sizeAutoradiography detects only labelled strandsCleaved fragments containing the control sequence show up on the gel from the samples without protein treatmentut are missing from those with protein treatment o The bound cognate protein blocked cleavageMissing bands on the fragment constitute the footprint General Principles of the Regulation of Transcription Initiation in Prokaryotes Transcription regulation goes in both directionsRegulator genes produce activator and repressor proteinsActivator and repressor proteins bind to specific regulator binding sites RBS next to the promoter of the gene being regulated o Promoter is where the RNA Pol binds Activators and repressors bind to a site nextthe promoterSmall molecule inducer and corepressors usually metabolites control binding of activatorrepressors to the RBS o An inducer makes an activator active A corepressor binds to a repressor makes the repressor bind to DNA which turns off transcriptionSwitching the tryptophan genes ON and OFFOperatora regulatory element within the promoterbetween 35 and 10 from the start siteTranscriptional repressornegatively regulates gene expressionIn the context of lac operon the cells want to express the lac operon in the presence of lactose but not in the absence of lactoseWhen theres a high level of tryptophan the cell doesnt express the tryptophan gene and the cell should be expressing the gene when theres low levels of tryptophanSigma 70 recognizes the whole promoter region The repressor is activein the presence
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