1 / 48

Eukaryotic RNA Polymerases

Gene Expression Eukaryotic Gene Transcription 9/18/08 Thomas Ryan, Ph.D. Biochemistry and Molecular Genetics tryan@uab.edu. Eukaryotic RNA Polymerases. Three DNA dependent RNA polymerases: RNA Pol I, II, and III All 3 are big, multimeric proteins (500-700 kD)

marsha
Download Presentation

Eukaryotic RNA Polymerases

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Gene ExpressionEukaryotic Gene Transcription9/18/08Thomas Ryan, Ph.D. Biochemistry and Molecular Geneticstryan@uab.edu

  2. Eukaryotic RNA Polymerases • Three DNA dependent RNA polymerases: RNA Pol I, II, and III • All 3 are big, multimeric proteins (500-700 kD) • All have 2 large subunits with sequences similar to  and ' in E. coli RNA polymerase, so catalytic site may be conserved • All interact with general transcription factors-GTFs • RNA Pol II is most sensitive to -amanitin

  3. RNA Polymerase II Inhibitor a-Amanitin “The Destroying Angel” - Amanita phalloides pol II >> pol III >>> pol I • Bicyclic octapeptide • Blocks elongation

  4. Yeast RNA Polymerase II Subunits

  5. Transcription Factors • The three RNA polymerases (I, II and III) interact with their promoters via protein:protein and protein:DNA interactions • These proteins or transcription factors (TFs) recognize and initiate transcription at specific promoter sequences • Some transcription factors (TFIIIA and TFIIIC for RNA polymerase III) bind to specific recognition sequences within the coding region

  6. Helix-Turn-Helix Motif

  7. Zinc-Finger Motif: C2H2 Class

  8. Basic Region-Leucine Zipper Motif: bZIP

  9. bZIP Transcription Factor

  10. General Transcription Factors (GTFs) • GTFs position RNAPs at transcription initiation sites, forming the preinitiation complex (PIC) • Transcription-initiation complex = RNAP + general transcription factors (GTF) bound to promoter region • Many of the GTFs that associate with RNAP II initiate transcription from TATA box-containing promoters have been identified • TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH • TFII = “transcription factor RNAP II”

  11. General Transcription Factors • TFIID is largest and consists of a TATA-box binding protein (TBP) and 8-10 TBP-associated factors (TAFIIs) • TBP is a “universal transcription factor” – associates with promoters of all three RNAPs, and promoters with and without a TATA box • Binding of TAFIIs extend the interactions of TFIID • TFIID has two roles: • foundation for the transcriptional PIC complex • Prevents nucleosome stabilization in the promoter region (antagonist to H1)

  12. TBP is used by all 3 RNA polymerases • TBP is a subunit of an important GTF for each of the 3 RNA polymerases: • TBP or TFIID for Pol II • SL1 for Pol I • TFIIIB for Pol III • It does NOT always bind to TATA boxes; promoters for RNA Pol I and Pol III (and even some for Pol II) do not have TATA boxes, but TBP is still used. • The GTFs that contain TBP may serve as positioning factors for their respective polymerases.

  13. Yeast TATA Binding Protein: TBP TBP binds in the minor groove of DNA TBP binding bends the DNA

  14. Promoters for RNAP I • RNA Pol I transcribes genes for the large rRNA precursor • There are hundreds of similar copies of this gene in each genome • RNA Pol I promoters (called class I) have two components: • Upstream control element: –156 to -107 • Core element: –45 to +20 • Two different transcription factors bind these sequences co-operatively: SL1 and UBF (TBP is a component of SL1)

  15. Promoters for RNAP III • Transcribes various small RNAs: 5S rRNA, tRNA precursors, U6 snRNA, etc • The 5S rRNA and tRNA genes have class III promoters • U6 snRNA and 7SL RNA gene have promoters that resemble RNAP II promoters • The 5S rRNA promoters are entirely within the coding region of the gene • The tRNA promoters contain two elements • The other promoters for U6 snRNA etc appear similar to RNAP II promoters (have TATA boxes) but -amanitin test indicates these are transcribed by RNAP III.

  16. PIC Assembly for RNA Pol III Genes TBP TBP TBP TBP

  17. RNA Polymerase II General Transcription Factors FactorSubunitsFunction TFIID - TBP 1 Recognize core promoter (TATA), Recruit TFIIB TFIID - TAFs 12 Recognize core promoter (non-TATA), Pos and Neg regulatory functions, HAT activity TFIIA 3 Stabilize TFIID and promoter binding TFIIB 1 Recruit RNA Pol II / TFIIF, Start site selection TFIIF 2 Assist RNA Pol II to bind promoter RNA Pol II 12 Enzymatic synthesis of RNA, Recruit TFIIE TFIIE 2 Recruit TFIIH, Modulate TFIIH helicase, ATPase, and kinase activities TFIIH 9 Promoter melting using helicase, Promoter clearance via CTD phosphorylation Modified from Roeder, R.G., Trends in Biochem. Sci. 21:327-334

  18. TBP-associated Factors (TAFs or TAFIIs) • Important for helping TBP to bind to promoters that lack TATA boxes. • There are different TAFs in different cells! • In vivo these factors are associated with additional proteins forming a larger complex of about 50 polypeptides. • It is hypothesized that this high MW complex may preassemble and interact with promoters in a single step.

  19. Eukaryotic RNA Pol II Transcription Formation of the PIC • TFIID binds TATA box via TBP subunit • TFIIA facilitates and stabilizes binding of TFIID complex

  20. Eukaryotic RNA Pol II Transcription Formation of the PIC • TFIIB binds to TFIID • TFIIB is a monomeric protein • C terminal domain contacts DNA and TBP • N terminal domain extends towards start site • Proximity of certain promoter- and enhancer-transcription factors (important for developmental regulation- their activation domain binds directly to TFIIB) • TFIIH close as well at this point, but not yet bound

  21. Eukaryotic RNA Pol II Transcription Formation of the PIC • TFIIF binds to RNAPII (preformed complex) – directs RNAPII to promoter • Binding of TFIIE to TFIIF/RNAPII complex and already positioned TFIIB helps positioning the RNAPII over start site: • Two large subunits of RNAP II interact with promoter DNA: CTD tail (unphsphorylated form) of RNAPII is in direct contact with TFIID • TFIIE is DNA-dependent ATPase- probably necessary for generating the energy for transcription

  22. Eukaryotic RNA Pol II Transcription Formation of the PIC • Binding of RNAP II/TFIIF/TFIIE to promoter activates TFIIH • TFIIH contains nine subunits • It has helicase activity- unwinds DNA downstream from the initiator site in the presence of ATP necessary for promoter clearance • It has protein kinase activity- phosphorylation of CTD tail of RNAPII • Phosphorylation detaches RNAPII from TFIID • Beginning of transcription by RNAPII

  23. Carboxyl-Terminal Domain (CTD Tail) • Stretch of 7 amino acids that is repeated multiple times (26-52 times): Tyr-Ser-Pro-Thr-Ser-Pro-Ser • Critical for viability • CTD tail becomes phosphorylated on ser and some tyr residues as the RNAP transcribes away from the promoter

  24. RNA Pol II Promoters Consist of two parts: Core promoter: - TATA box (position at ~ -30) - initiator (on the transcription start site) 2. Proximal Promoter Elements (can be upstream, downstream or internal) Upstream Element TATA Initiator Downstream Element

  25. Eukaryotic TATA Box TATA motif is usually located at position -25 Consensus Sequence

  26. Eukaryotic Promoter Regions Initiator

  27. RNA Polymerase II Promoter Consensus Sequences of Transcription Factor Binding Sites

  28. Proximal Control Elements of Genes Modular Factor Binding Sites

  29. Coordinate Regulation Via Response Elements • Multiple genes are transcribed in response to different cues: for example, heat shock, hormone levels, developmental events, phorbol esters, heavy metals, metabolite concentrations, etc. • Similarly responsive genes will have a DNA sequence located in cis to the gene called a response element. • These response elements are binding sites for transacting factors that are activated in response to the environmental cue. • The location of these elements relative to the start site of transcription is not conserved between genes: eg. a cis element that leads to transcription in response to a hormone may be located at –300 in one gene and –175 in another

  30. Response Elements: Coordinate Regulation Metallothionein Gene Promoter

  31. Coordinate Regulation by Hormones/Steroid Receptors Adapted from Molecular Biology of the Cell, 4th Edition

  32. Steroid Receptors Upon binding to the hormone cortisol, the cytoplasmic glucocorticoid receptor displaces an inhibitory protein (Hsp90) and moves to the nucleus where is can interact with glucocorticoid response elements (GREs) in the DNA affecting gene transcription. Adapted from Molecular Biology of the Cell, 4th Edition

  33. Activation of Transacting Factors

  34. Control of Cellular Differentiation By TFs

  35. Enhancers • Control elements that stimulate transcription • Bind multiple different transcription factors • Transcription factors that recognize enhancer = activators or enhancer binding proteins • Activators interact with general transcription factors • Negative enhancer is a silencer

  36. Enhancers • Stimulate expression of genes over long disances (up to 50kb) • Occur upstream, downstream, in introns or in exons • Orientation independent • May be cell-type specific

  37. Enhancers: Action at a Distance Activation of transcription initiation in eukaryotes by recruitment of the eukaryotic RNA polymerase II holoenzyme complex (100 protein subunits).

  38. Insulators / Boundary Elements Boundary elements block encroachment of heterochromatin from neighboring loci. They also stop the unregulated enhancement or activation of neighboring genes outside of their chromosomal domain.

  39. Hypomethylation (Active) Vs. Hypermethylation (Silenced)

  40. Silencing: Histone Deacetylation HDAC • Some repressors recruit histone deacetylase, which removes acetyl groups from histones resulting in gene silencing

  41. Histone Acetylation and Deacetylation Histone acetyl transferases HAT HDAC Histone deacetylases

  42. Histone Code Histone tails are postranslationally modified by acetylation, methylation, phosphorylationand ubiquitination. These modifications have a profound effect on gene activity. The specific set of modifications is termed the “histone code” .

  43. Chromatin Remodeling Remodeling complexes allow access of replication and transcription factors to the DNA. Remodeling requires ATP

  44. Gene Activation By Chromatin Remodeling Transcription Factor Access

  45. HATs open chromatin Nucleosome remodelersTranscription Factor Coactivators

  46. Assembly of preinitiation complex on open chromatin

  47. Chromatin remodeling of diploid somatic cell nucleus in the egg cytoplasm reprograms the nucleus to recapitulate development.

More Related