Molecular biology - Chapter 10: Eukaryotic rna polymerases and their promoters

Molecular BiologyFourth 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 Three Nuclear PolymerasesEukaryotic nuclei contain three RNA polymerasesThese can be separated by ion-exchange chromatographyRNA polymerase I found in nucleolusLocation suggests in 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 enzymeTechnique to help determine whether a polypeptide copurifies or is a subunit is called epitope tagging6Epitope TaggingAdd an extra domain to one subunitOther subunits normalPolymerase labeled by growing in labeled amino acidsPurify with antibodyDenature with detergent and separate on a gel7Polymerase IIOriginal 10 subunits are placed in 3 groups:Core – related in structure and function to bacterial core subunitsCommon – found in all 3 nuclear RNA polymerasesNonessential subunits – conditionally dispensable for enzymatic activity8Core SubunitsThree polypeptides, Rpb1, Rpb2, Rpb3 are absolutely required for enzyme activityThese are homologous to b’-, b-, and a-subunitsBoth Rpb1 and b’-subunit binds DNARpb2 and b-subunit are at or near the nucleotide-joining active siteRpb3 does not resemble a-subunitThere is one 20-amino acid subunit of great similarity2 subunits are about same size, same stoichiometry2 monomers per holoenzyme9Common SubunitsThere are five common subunitsRpb5Rpb6Rpb8Rpb10Rpb12Little known about functionThey are all found in all 3 polymerasesSuggests play roles fundamental in transcription10Subunits Nonessential for ElongationRpb4 and Rpb7 Dissociate fairly easily from polymeraseFound in substoichiometric quantitiesMight shuttle from one polymerase II to anotherRpb4 may help anchor Rpb7 to the enzymeMutants without Rpb4 and Rpb7 transcribes well, but cannot initiate at a real promoterRpb7 is an essential subunit, so must not be completely absent in the mutant11Heterogeneity 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 ser in the repeating heptad of the CTDEnzyme with IIa binds to the promoterEnzyme with IIo is involved in transcript elongation12The Three-Dimensional Structure of RNA Polymerase IIStructure of yeast polymerase II (specifically pol II 4/7) at atomic resolution reveals a deep cleft that accepts a linear DNA template from one end to anotherCatalytic center lies at the bottom of the cleft and contains a Mg2+ ionA second Mg2+ ion present in low concentrationsGeometry allows enough space for: TFIID to bind at the TATA box of the promoterTFIIB to link the polymerase to TFIIDPlaces polymerase correctly to initiate transcription133-D Structure - 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 has shifted closed over the DNA and RNAClosed clamp ensures that transcription is processive – able to transcribe a whole gene without falling off and terminating prematurely14Position of Nucleic Acids in the Transcription BubbleDNA template strand is shown in blueDNA nontemplate strand shown in greenRNA is shown in red15Position 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 DNA16Proposed 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 transcription173-D Structure - RNA Polymerase II in the Posttranslocation StateX-ray crystallography has shown the lid of Rpb1 interacts with the DNA-RNA hybrid to force the hybrid open after base pair -8The lid then interacts with bases of the nascent RNA to keep the hybrid melted beyond base pair -8The rudder of Rpb1 collaborates with lid to keep the hybrid melted by interacting with bases -9 and -10Fork loop 1 of Rpb2 interacts with bases -5, -6, and -7 of the RNA to keep the RNA-DNA hybrid together18Structural 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 overlapRotation of nucleotide between the E and A sites may play a role in base and sugar specificityTwo 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 nucleotide19The Role of Rpb4 and Rpb7Structure of the 12-subunit RNA polymerase II reveals that, with Rpb4/7 in place, clamp is forced shutInitiation occurs, with its clamp shut, it appears that the promoter DNA must melt to permit template DNA strand to enter the active siteThe Rpb4/7 extends the dock region of the polymerase, which makes binding of transcription factors easier2010.2 PromotersThree eukaryotic RNA polymerases have:Different structuresTranscribe different classes of genesExpect that the 3 polymerases would recognize different promoters21Class II PromotersPromoters recognized by RNA polymerase II (class II promoters) are similar to prokaryotic promotersConsidered to have two parts:Core promoter having 4 elementsUpstream promoter element22Core Promoter Elements – TATA BoxTATA box Found on the nontemplate strandVery similar to the prokaryotic -10 boxThere are frequently TATA-less promotersHousekeeping genes that are constitutively active in nearly all cells as they control common biochemical pathwaysDevelopmentally regulated genes 23Linker ScanningSystematically substitute a 10-bp linker for 10-bp sequences throughout the promoterFound that mutations within the TATA box destroyed promoter activity24Core Promoter ElementsIn addition to TATA box, core promoters are:TFIIB recognition element (BRE)Initiator (Inr)Downstream promoter element (DPE)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 Promoters for housekeeping genes tend to lack them25Upstream ElementsUpstream promoter elements are usually found upstream of class II core promotersDiffer from core promoters in binding to relatively gene-specific transcription factorsGC boxes bind transcription factor Sp1CCAAT boxes bind CTF (CCAAT-binding transcription factor)Upstream promoter elements can be orientation-independent, yet are relatively position-dependent26Class 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 important27Class III PromotersRNA polymerase III transcribes a set of short genesThese have promoters that lie wholly within the genesThere are 3 types of these promoters28Promoters of Some Polymerase III GenesType I (5S rRNA) has 3 regions:Box AShort intermediate elementBox CType II (tRNA) has 2 regions:Box A Box BType III (nonclassical) resemble those of type II2910.3 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 it30