Three types of RNA polymerase in eukaryotic nucleiSubunit composition of eukaryotic RNA polymerasesComparison of 3-D structures of bacterial and eukaryotic RNA polymerasesPowerPoint PresentationCore Promoter ElementsRNA Pol II is recruited to core promoter with the help from multiple general transcription factors. In vitro stepwise assembly of the RNA Pol II pre-initiation complex (PIC) at core promoter for basal transcriptionBasal (‘General’) Transcription Factors for RNA Polymerase IITBP (TATA-box binding protein)Eukaryotic transcription cycleSlide 10Cis-acting control elementsDNA affinity chromatography for purification of Sp1Analyses of affinity-purified Sp1 protein“Gel shift”: electrophoretic mobility shift assay (EMSA) for studying the interaction of Sp1 with GC-boxIn vitro transcription assay to measure Sp1 activityIn vivo assay for transcription factor activitySlide 17Slide 18Slide 19Slide 20Three types of RNA polymerase in eukaryotic nucleiType Location RNA synthesized Effect of -amanitinI Nucleolus Pre-rRNA for 18, 5.8 and 28S rRNAs InsensitiveII Nucleoplasm Pre-mRNA, some snRNAs Sensitive to 1 g/mlIII Nucleoplasm Pre-tRNAs, 5S rRNA, some snRNAs Sensitive to 10 g/ml(Also- Organelle RNAPs in Mitochondria and Chloroplasts. Encoded by organelle genomes. Similar to bacterial RNAPs.)• -amanitin from Amanita Phalloides binds tightly to RNA Pol II and blocks transcriptional elongation.• RNA Pol I transcribe 1 gene at ~200 copies. The gene for the 45S pre-rRNA is present in tandem array.• RNA Pol II transcribe ~25,000 genes;• RNA Pol III transcribe 30-50 genes at variable copy numbers.Subunit composition of eukaryotic RNA polymerases•All three yeast polymerases have five core subunits that exhibit some homology with the , ‘,  and  subunits in E. coli RNA polymerase. •RNA polymerases I and III contain the same two non-identical -like subunits, whereas polymerase II has two copies of a different -like subunit.•All three polymerases share four other common subunits. In addition, each RNA polymerase contains three to seven unique smaller subunits.•The largest subunit (1) of RNA polymerase II also contains an essential C-terminal domain (CTD). 27 (yeast) to 52 (human) copies of (YSPTSPS). •Phosphorylation of CTD is important for transcription and RNA processing.Comparison of 3-D structures of bacterial and eukaryotic RNA polymerases(subunits 4 and 7 are missing)Cyan: alternating -turns; Pink: extended regions.Core Promoter ElementsMany genes, which are transcribed at low rates (e.g. genes encoding the enzymes required for basic metabolic processes required in all cells, often called “housekeeping genes”) do not contain a TATA box or an initiator. Most genes of this type contain a CG-rich region, or CpG island, of 20-50 nucleotides within ~100 base pairs upstream from the start site. Transcription of these genes can begin at any one of multiple possible sites over an extended region.RNA Pol II is recruited to core promoter with the help from multiple general transcription factors.In vitro stepwise assembly of the RNA Pol II pre-initiation complex (PIC) at core promoter for basal transcriptionBasal (‘General’) Transcription Factors for RNA Polymerase IITotal: 43-44 polypeptides and over 2 million daltons.TBP (TATA-box binding protein)•Conserved C-terminal domain of 180 amino acids.•A monomer with a saddle-shaped structure; the two halves show an overall dyad symmetry but are not identical.•Binds multiple transcription factors (TAFs, TFIIB and TFIIA).•Binds in the minor groove and significantly bends DNA.Eukaryotic transcription cycleOnly the unphosphorylated RNA Pol II enters PIC.The TFIIH complex has both helicase and kinase activities that can unwind DNA and phosphorylate the CTD tail of RNA Pol II.Release of TFIIE and then IIH during the synthesis of the initial 60-70nt.CTDPIC assemblyPol IICTDPol II Pol II5555TFIIHPromoter clearance& pausing for cappingCycT1CDK9P-TEFbRelease from pausingPol IIProductive elongationPol IIPol IICTDCTDFCP1Recycling5’ cap255552222555522225555222RNA+1CTD heptapeptide repeats: 27-52 x (YS2PTS5PS)Phosphorylation states of Pol II CTD during transcription cycleCis-acting control elements(a) Genes of multicellular organisms contain both promoter-proximal elements and enhancers (collectively referred to as cic-acting control elements) in addition to core promoter element(s).(b) Enhancers function in a distance, position and orientation-independent manner. Long distance interactions are achieved by forming looped DNA.(c) Most yeast genes contain only one regulatory region, called an upstream activating sequence (UAS), and a TATA box, which is ≈90 base pairs upstream from the start site. (Also note: many yeast genes do not contain introns).(d) In multicellular organisms, one standard promoter-proximal element often located in the CpG island is a GC-box (GGGCGGGC) recognized by the ubiquitous transcriptional activator Sp1.DNA affinity chromatography for purification of Sp1Analyses of affinity-purified Sp1 proteinDNase I footprint on SV40 promoterSDS-PAGE/ silverstainSp1NaClLane 2 contains total cell proteins prior to affinity purification;Lanes 3&4 contain purified Sp1 protein washed off the affinity column.6 x GC-boxes“Gel shift”: electrophoretic mobility shift assay (EMSA) for studying the interaction of Sp1 with GC-boxFree DNA probe**Protein-DNA complex1. Prepare labeled DNA probe2. Bind protein3. Native gel electrophoresisAdvantage: sensitive Disadvantage: requires stable complex; little information about where protein is binding on DNAON: protein mixture loaded onto an ion-exchange column.Fraction 1-22: fractions eluted from the column with increasing salt concentrations.Sp1GC-boxIn vitro transcription assay to measure Sp1 activity•The adenovirus DNA template used here does not contain any Sp1-binding sites (GC-box) and is therefore used as a negative control.•In vitro transcription reactions contain template DNA, labeled ribonucleoside triphosphates, and purified general transcription factors and RNA Pol II. Purified Sp1 is added to the reactions indicated with “+”.In vivo assay for transcription factor activityco-transfection• Host cells should lack the gene encoding protein X and the reporter protein.• The production of reporter-gene RNA transcripts or the activity of the encoded protein can be assayed.• If reporter-gene transcription is