Bài giảng Molecular Biology - Chapter 10 Eukaryotic RNA Polymerases and Their Promoters

Molecular BiologyFifth EditionChapter 10Eukaryotic RNA Polymerases and Their PromotersLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.110.1 Multiple Forms of Eukaryotic RNA PolymeraseThere are at least two RNA polymerases operating in eukaryotic nucleiOne transcribes major ribosomal RNA genesOne or more to transcribe rest of nuclear genesRibosomal genes are different from other nuclear genesDifferent base composition from other nuclear genesUnusually repetitiveFound in different compartment, the nucleolus2Separation of the 3 Nuclear PolymerasesEukaryotic nuclei contain three RNA polymerasesThese can be separated by ion-exchange chromatographyRNA polymerase I found in nucleolusLocation suggests it transcribes rRNA genesRNA polymerases II and III are found in the nucleoplasm3Roles of the Three RNA PolymerasesPolymerase I makes large rRNA precursorPolymerase II makes Heterogeneous nuclear RNA (hnRNA)small nuclear RNAPolymerase III makes precursors to tRNAs, 5S rRNA and other small RNA4RNA Polymerase Subunit Structures5Polymerase II StructureFor enzymes like eukaryotic RNA polymerases, can be difficult to tell: Which polypeptides copurify with polymerase activity Which are actually subunits of the enzymeEpitope tagging is a technique to help determine whether a polypeptide copurifies or is a subunit6Epitope TaggingAdd an extra domain to one subunit of RNA polymeraseOther subunits normalImmunopreciptate with antibody directed against epitopeDenature with SDS detergent and separate via electrophoretic gel7Core Subunits of RNA PolymeraseThree polypeptides, Rpb1, Rpb2, Rpb3 are absolutely required for enzyme activity (yeast)Homologous to b’-, b-, and a-subunits (E.coli)Both Rpb1 and b’-subunit binds DNARpb2 and b-subunit are at or near the nucleotide-joining active siteSimilarities between Rpb3 and a-subunitThere is one 20-amino acid subunit of great similarity2 subunits are about same size, same stoichiometry2 monomers per holoenzymeAll above factors suggest they are homologous8Common SubunitsThere are five common subunitsRpb5Rpb6Rpb8Rpb10Rpb12Little known about functionThey are all found in all 3 polymerases which suggests they play roles fundamental to the transcription process9SummaryThe genes encoding all 12 RNA polymerase II subunits in yeast have been sequenced and subjected to mutational analysisThree of the subunits resemble the core subunits of bacterial RNA polymerases in both structure and functionFive are found in all three nuclear RNA polymerases, two are not required for activity and two fall into none of these categories10Heterogeneity of the Rpb1 SubunitRPB1 gene product is subunit IISubunit IIa is the primary product in yeastCan be converted to IIb by proteolytic removal of the carboxyl-terminal domain (CTD) which is 7-peptide repeated over and overConverts to IIo by phosphorylating 2 serine in the repeating heptad of the CTDEnzyme with IIa binds to the promoterEnzyme with IIo is involved in transcript elongation11The Three-Dimensional Structure of RNA Polymerase IIStructure of yeast polymerase II (pol II 4/7) reveals a deep cleft that accepts a DNA templateCatalytic center lies at the bottom of the cleft and contains a Mg2+ ionA second Mg2+ ion is present in low concentration and enters the enzyme bound to each substrate nucleotide123-D Structure of RNA Polymerase II in an Elongation ComplexStructure of polymerase II bound to DNA template and RNA product in an elongation complex has been determinedWhen nucleic acids are present, the clamp region of the polymerase is closed over the DNA and RNAClosed clamp ensures that transcription is processive – able to transcribe a whole gene without falling off and terminating prematurely13Position of Nucleic Acids in the Transcription BubbleDNA template strand is shown in blueDNA nontemplate strand shown in greenRNA is shown in red14Position of Critical Elements in the Transcription BubbleThree loops of the transcription bubble are:Lid: maintains DNA dissociationRudder: initiating DNA dissociation Zipper: maintaining dissociation of template DNA15Proposed Translocation MechanismThe active center of the enzyme lies at the end of pore 1Pore 1 also appears to be the conduit for: Nucleotides to enter the enzymeRNA to exit the enzyme during backtrackingBridge helix lies next to the active centerFlexing this helix may function in translocation during transcription16Structural Basis of Nucleotide SelectionMoving through the entry pore toward the active site of RNA polymerase II, incoming nucleotide first encounters the E (entry) siteE site is inverted relative to its position in the A site (active) where phosphodiester bonds formE and A sites partially overlapTwo metal ions (Mg2+ or Mn2+) are present at the active siteOne is permanently bound to the enzymeThe other enters the active site complexed to the incoming nucleotide17The Trigger LoopIn 2006 a crystal structure with GTP rather than UTP in the A site, opposite a C, revealed a part of Rpb1 roughly encompassing residues 1070 to 1100 - a trigger loopThe trigger loop only comes into play when the correct substrate occupies the A site and makes several important contacts with the substrate that presumably stabilize the substrates association with the active site and contribute to the specificity of the enzyme18The Role of Rpb4 and Rpb7Structure of the 12-subunit RNA polymerase II reveals that, with Rpb4/7 in place, the clamp is forced shutInitiation occurs, with its clamp shut, it appears that the promoter DNA must melt to permit the template DNA strand to enter the active siteThe Rpb4/7 extends the dock region of the polymerase, making it easier for certain general transcription factors to bind, thereby facilitating transcription initiationRpb7 can bind to nascent RNA and may direct it toward the CTD1910.2 PromotersThree eukaryotic RNA polymerases have:Different structuresTranscribe different classes of genesWe would expect that the three polymerases would recognize different promoters20Class II PromotersClass II promoters are recognized by RNA polymerase IIConsidered to have two parts:Core promoter - attracts general transcription factors and RNA polymerase II at a basal level and sets the transcription start site and direction of transcriptionProximal promoter - helps attract general transcription factors and RNA polymerase and includes promoter elements upstream of the transcription start site21Core Promoter Elements – TATA BoxTATA box Very similar to the prokaryotic -10 boxPromoters have been found with no recognizable TATA box that tend to be found in two classes of genes:1 - Housekeeping genes that are constitutively active in nearly all cells as they control common biochemical pathways2 - Developmentally regulated genes 22Core Promoter ElementsThe core promoter is modular and can contain almost any combination of the following elements:TATA box TFIIB recognition element (BRE)Initiator (Inr)Downstream promoter element (DPE)Downstream core element (DCE)Motif ten element (MTE)At least one of the four core elements is missing in most promotersTATA-less promoters tend to have DPEsPromoters for highly specialized genes tend to have TATA boxes 23ElementsPromoter elements are usually found upstream of class II core promotersThey differ from core promoters in binding to relatively gene-specific transcription factorsUpstream promoter elements can be orientation-independent, yet are relatively position-dependent24Class I PromotersClass I promoters are not well conserved in sequence across speciesGeneral architecture of the promoter is well conserved – two elements:Core element surrounding transcription start siteUpstream promoter element (UPE) 100 bp farther upstreamSpacing between these elements is important25Class III PromotersRNA polymerase III transcribes a variety of genes that encode small RNAsThe classical class III genes have promoters that lie wholly within the genesThe internal promoter of the type I class III gene is split into three regions: box A, a short intermediate element and box CThe internal promoters of the type II genes are split into two parts: box A and box BThe promoters of the nonclassical class III genes resemble those of class II genes26Promoters of Some Polymerase III GenesType I (5S rRNA) has 3 regions:Box A, Short intermediate element, and Box CType II (tRNA) has 2 regions:Box A and Box BType III (nonclassical) resemble those of type II2710.3 Enhancers and SilencersThese are position- and orientation-independent DNA elements that stimulate or depress, respectively, transcription of associated genesAre often tissue-specific in that they rely on tissue-specific DNA-binding proteins for their activitiesSome DNA elements can act either as enhancer or silencer depending on what is bound to it28EnhancersEnhancers act through the proteins that are bound to them, enhancer-binding proteins or activatorsThese proteins appear to stimulate transcription by interacting with other proteins called general transcription factors at the promoter that promote the formation of a preinitiation complexEnhancers are frequently found upstream of the promoter they control although this is not an absolute rule29SilencersSilencers, like enhancers, are DNA elements that can act at a distance to modulate transcription but they inhibit, rather than stimulate, transcriptionIt is thought that they work by causing the chromatin to coil up into a condensed, inaccessible and inactive form thereby preventing the transcription of neighboring genes30Vital themeThe finding that a gene is much more active in one cell type than another leads to an extremely important point: All cells contain the same genes, but different cell types differ greatly from one another due to the proteins expressed in each cellThe types of proteins expressed in each cell type is determined by the genes that are active in those cellsPart of the story of the control of gene expression resides in the expression of different activators in different cell types that turn on different genes to produce different proteins31