lec 1 notes.docx

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University of Toronto St. George
Stavroula Andreopoulos

TRANSCRIPTION I PROKARYOTES lec 1ProkaryotesEukaryotesOnly has 1 RNAP to bind promoter for Has 3 different RNAPs IIIIII to transcribe initiationRNAsPrimary transcript of mRNA modification has Primary RNA transcripts are extensively little to no modification tRNA and rRNA modified eg Splicing cappingtranscripts undergo modificationPrimary RNA transcript serves as mRNA and Highly regulated by the binding of is elongated Activationrepression done by Transcription factors TFsregulatory proteins eg Lac operonTranscription is slower at 50ntsTranscription rate is 80ntsSimultaneous transcription and translation so Transcripts are exported out of nucleus into no spatial or temporal differencescell cytoplasmNot need TFs for transcription only to TFs are needed for transcriptionrecognise the promoter sequenceTermination by RNAP at the end of the geneTermination is more complexSimilarities 1 Overall RNAP quite similar between prok and euk RNA synthesised de novo as opposedto DNA2 RNA synthesis is in 3 stages initiation elongation and termination ie nascent RNA 53 synthesis from Nterminus Cterminus as it is complementary to the DNA template RNAP moves 3 to 5 on the DNA template noncodingantisense strand to add incoming nt to 3OH and release PPi to drive the reaction forwardRNAP general functionsaFinds cisacting elements at promoterinitiation sites of DNA so does not need a primerFOOTPRINTING determines where specific DBP DNA binding protein latches on to DNA strand32iAdd P radiolabel to 5 end of dsDNARNAPDNase IiiDNase I creates 1 nick at a time to denature dsDNA and get radiolabelled fragments of various sizesiiiLabelle fragments go through gel electrophoresis to determine DBP location of DNA ie strands absentpromoter regionivCompare to naked dsDNA with no DBP which has no nicks so all strands present5bUnwinds dsDNA but no proofreading so has an error rate of 10 but can tolerate this since many transcripts are madecUnidirectional and processive so there is correct ribonucleoside triphosphate selectiondTerminationeActivationrepression is done by TFs in euks and by proteins in proksfFundamental phosphodiester reactionPurinesglycosidic linkage on N9Pyrimidinesglycosidic linkage on N1 Ecoli RNAP structure400kDa with core enzyme consisting of 4 subunits ieit has the 2lowest dissociation constant10Holoenzyme is a pentamer ofwheredecreases DNA binding 2affinity of RNAPis released after 10nts are synthesised in order to bind to another core complex it is catalyticActive site is similar to that of DNAP but it has 3 sites that allow Hydrogen bond formation between ribonts ie AS preinsertion and insertion sitesst1Growing RNA chain has its 1 nt in the AS which has an 2Mg to coordinate 3 Asp residues22Incoming nt enters via the insertion site with its own Mg3Catalysis in AS allows 3OH of growing chain to attach the Phosphate of the incoming chainrelease of PPi24Incoming chains Mg leaves with PPi1 TRANSCRIPTION INITIATIONrapid random walk by continuing outwards until it reaches 1011a promoter sequence so does not jump on and offRate binding of 10 Ms
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