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Berkeley MCELLBI 110 - Lecture Notes

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Steps in geneexpression:comparison ofprokaryotic andeukaryotic cellsSix steps at which eukaryotic gene expression can becontrolled.In prokaryotic cells, genes do not have introns (no step 2)and transcription and translation are not separated in spaceand time (no step 3).12Transcription controlDNARNAProcessing RNATransport3Translation Control4 mRNADegradation5ProteinActivity6Primary RNATranscriptRNAmRNAInactive mRNAProteinInactiveProteinNucleusCytoplasmTranscription of DNA into RNA by RNApolymerase.1. Requires DNA template,4 ribonucleotide 5’ triphosphates, Mg+2.2. De novo synthesis: does not require a primer.Low fidelity compared to DNA polymerase:errors 1/104-105. RNA polymerase incorporates~30 nt/s (much slower than DNA polymerase).3. Activity highly regulated in vivo: at initiation,elongation and termination.4. The nucleotide at the 5’ end of an RNA strandretains all three of its phosphate groups; allsubsequent nucleotides release pyrophosphate(PPi) when added to the chain and retain onlytheir a phosphate (red).5. The released PPi is subsequently hydrolyzedby pyrophosphatase to Pi, driving theequilibrium of the overall reaction toward chainelongation.6. In most cases, only one DNA strand istranscribed into RNA.Template strandNon-template (coding) strandTranscription of two genes on different template strands. Note thatgrowth of the transcript always occurs in the 5’-to-3’ direction.The convention for designating the 5’ and 3’ ends of a geneBiochemical studies of bacterial RNA polymerase1. DNA binding assays- DNaseI footprintingGel shift2. Role of individual subunits- dissociation of holoenzyme by column chromatographyDNase I footprinting, a common techniquefor identifying protein-binding sites in DNA. 1. A DNA fragment is labeled at one end with32P (red dot).2. Portions of the sample then are digestedwith DNase I in the presence and absence of aprotein that binds to a specific sequence in thefragment.3. A low concentration of DNase I is used sothat on average each DNA molecule is cleavedjust once (vertical arrows).4. The two samples of DNA then areseparated from protein, denatured to separatethe strands, and electrophoresed. The resultinggel is analyzed by autoradiography, whichdetects only labeled strands and revealsfragments extending from the labeled end tothe site of cleavage by DNase I.DNaseI footprint of RNAP on the lacpromoterLanes 1 and 2 are DNA sequencing reactions for orientationLane 3 is the no protein controlLane 4 contained RNAPFootprint:Advantage- can assay short-lived interactions; proteins have verycharacteristic binding patternsDisadvantage- requires nearlystoichiometric binding“Gel shift”: electrophoretic mobility shift assay(“EMSA”) for DNA-binding proteinsFree DNA probe**Protein-DNA complex1. Prepare labeled DNA probe2. Bind protein3. Native gel electrophoresisAdvantage: sensitive Disadvantage: requires stable complex; little “structural” information about which protein is bindingIon-exchange chromatographyDissociation of RNAP and purification of sby ion-exchange chromatographyaab’bsw[NaCl][protein]Fraction numbersaab’bwCarboxymethyl- (-CO2-2) or phospho- (-PO3-2) celluloseE. coli RNA polymerase holoenzyme bound to DNA.Subunit Stoichiometry Role in holoenzymea 2 Binds regulatory sequences/proteinsb 1 Polymerase; Binds DNA templateb’ 1 Polymerase; Binds DNA templates 1 Promoter recognitionw 1 RNAP assemblywThermus aquaticus core RNA polymerasePDB (Protein Data Bank)http://www.rcsb.org/pdb/File 1HQMMinakhin, L. et al. Proc.Nat.Acad.Sci.USA 98 pp. 892 (2001)a Blueb Redb’ Greenw OrangeA single RNA polymerase makesmultiple types of RNAs inprokaryotesThe dissociable sigma subunit gives promoter specificity toprokaryotic RNA polymerase (RNAP)aab’bsCore enzyme+aab’bsHoloenzymeNo specific promoter binding; tight non-specific DNA binding (Kd ~5 x 10-12 M)Specific promoter binding; weak non-specific DNA binding (Kd ~10-7 M)wwTranscription initiation by prokaryotic RNA polymerasesCore enzymeHoloenzymeaab’bsPromoter-35-10“scanning”Open complex; initiationaab’bsClosed complexrNTPsPPiaab’b5’pppAmRNASigma Factors of E. coliSigmaFactor Promoters Recognized Promoter Consensus-35 Region -10 Regions70 Most genes TTGACAT TATAATs32 Genes induced by heat shock TCTCNCCCTTGAA CCCCATNTAs28 Genes for motility and chemotaxis CTAAA CCGATATs38 Genes for stationary phase and stress response ? ?-24 Region -12 Regions54 Genes for nitrogen metabolism and other functions CTGGNA TTGCATranscriptional elongationSupercoiling of DNA during transcription causes arequirement for topoisomerasesRho-independent prokaryotic transcription terminationRho-dependent transcription terminationPlatt, Ann. Rev. Biochem. 55: 339 (1986)Rho binding siteterminationRho: forms RNA-dependent hexameric ATPase, translocates along RNA 5’-to-3’, promoting dissociation from


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Berkeley MCELLBI 110 - Lecture Notes

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