Chromatographic Separation of the three forms of RNA Polymerase II.

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1 Chromatographic Separation of the three forms of RNA Polymerase II.

2 α-amanitin

3 α-amanitin bound to Pol II

5 Function of the three enzymes.

6 Yeast Pol II.

7 RNA Polymerase Subunit Structures 10-7

8 Subunit structure.

9 Core Subunits of RNA Polymerase Three polypeptides, Rpb1, Rpb2, Rpb3 are absolutely required for enzyme activity (yeast) Homologous to β -, β-, and α-subunits (E.coli) Both Rpb1 and β -subunits bind DNA Rpb2 and β-subunits are at or near the nucleotidejoining active site Similarities between Rpb3 and α-subunit There is one 20-amino acid segment of great similarity 2 subunits are about same size, same stoichiometry 2 monomers per holoenzyme All above factors suggest they are homologous 10-9

10 Comparison of yeast RNA polymerase II with E. coli core and holoenzyme Pol II Core Holo

11 Epitope-tagging

12 Epitope-tagged purification of RNA polymerase II from yeast.

14 Heterogeneity of the Rpb1 Subunit RPB1 gene product is subunit IIa Subunit IIa is the primary product in yeast Can be converted to IIb by proteolytic removal of the carboxyl-terminal domain (CTD) which is 7-peptide repeated over and over Converts to IIo by phosphorylating serine 2 in the repeating heptad of the CTD Enzyme with IIa binds to the promoter Enzyme with IIo is involved in transcript elongation 10-14

16 The Three-Dimensional Structure of RNA Polymerase II Structure of yeast polymerase II (pol II Δ4/7) reveals a deep cleft that accepts a DNA template Catalytic center lies at the bottom of the cleft and contains a Mg 2+ ion A second Mg 2+ ion is present in low concentration and enters the enzyme bound to each substrate nucleotide 10-16

18 Structure of elongation complex

19 Model of Yeast RNA Polymerase II

20 Position of Nucleic Acids in the Transcription Bubble DNA template strand is shown in blue DNA nontemplate strand shown in green RNA is shown in red 10-20

21 Position of Critical Elements in the Transcription Bubble Three loops of the transcription bubble are: Lid: maintains DNA dissociation Rudder: initiating DNA dissociation Zipper: restoring ds DNA 10-21

22 Possible translocation mechanism

23 Matched vs mismatched NTP at active site

24 Matched vs mismatched trigger loop conformations

27 Class II Promoters Class II promoters are recognized by RNA polymerase II Considered 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 transcription Proximal promoter - helps attract general transcription factors and RNA polymerase and includes promoter elements upstream of the transcription start site 10-27

28 Core Promoter Elements TATA Box TATA box Very similar to the prokaryotic -10 box Promoters 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 pathways 2 - Developmentally regulated genes 10-28

29 Core Promoter Elements The 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 promoters TATA-less promoters tend to have DPEs Promoters for highly specialized genes tend to have TATA boxes 10-29

30 Elements Promoter elements are usually found upstream of class II core promoters They differ from core promoters in binding to relatively gene-specific transcription factors Upstream promoter elements can be orientation-independent, yet are relatively position-dependent 10-30

31 In vitro transcription assays run-offs primer extension GC-less cassette

32 Fractionation of Pol II GTFs from HeLa cells

33 DABF Pol II complex

34 Structure of TBP bound to DNA.

35 TAFs.

36 TAF comparison.

37 TAF activity

38 In vivo Dependence on TAFs.

39 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. The Drosophila tudor gene has two promoters one uses TBP and the other TRF-1

40 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

41 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

42 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

43 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

44 10.3 Enhancers and Silencers These are position- and orientationindependent DNA elements that stimulate or depress, respectively, transcription of associated genes Are often tissue-specific in that they rely on tissue-specific DNA-binding proteins for their activities Some DNA elements can act either as enhancer or silencer depending on what is bound to it 10-44

45 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

46 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

47 Fractionation of Pol II GTFs from HeLa cells

48 RNA polymerase II general transcription factors 6 general transcription factors have been identified and purified: IIA, IIB, IID, IIE, IIF and IIH

49 TFIIA Yeast 32kD, 13.5kD Human and fly 30kD, 30kD, and 13kD Binds to IID and stabilizes protein-dna interaction

50 TFIIB Yeast 26kD Humans 35kD Single subunit allows polymerase to associate with the DAB complex Orients polymerase and determines the direction of transcription

51 Structure and Function of TFIIB Structural studies have revealed that TFIIB binds to TBP at the TATA box via its C- terminal domain and polymerase II via its N- terminal domain The protein provides a bridging action that effects a coarse positioning of polymerase active center about bp downstream of the TATA box Plays an important role in establishing the transcription start site 11-51

53 TFIIF Yeast 105kD, 55kD, 30kD Humans 70kD, 30kD Binds to RNA polymerase and interacts with TFIIB in the DAB complex Binds close to the active center of Pol II and the N-terminus of TFIIB

54 DABF Pol II complex

55 TFIIH TFIIH is the last general transcription factor to join the preinitiation complex (contains 9 subunits) Separates into 2 complexes Protein kinase complex of 4 subunits Core TFIIH complex of 5 subunits with 2 DNA helicase/ ATPase activities Plays two major roles in transcription initiation: Phosphorylates the CTD of RNA polymerase II Unwinds DNA at the transcription start site to create the transcription bubble 11-55

57 Phosphorylation of the CTD of RNA Polymerase II The preinitiation complex forms with hypophosphorylated form of RNA polymerase II (IIA) Then TFIIH phosphorylates serine 5 in the heptad repeat in the carboxyl-terminal domain (CTD) of the largest RNA polymerase subunit This creates the phosphorylated form of the polymerase enzyme (IIO) This phosphorylation is essential for initiation of transcription 11-57

58 Phosphorylated Polymerase IIO During Elongation During the shift from initiation to elongation, two serines of the CTD are phosphorylated (serines 2 and 5 - and sometimes serine 7) Evidence exists that transcription complexes near the promoter have CTDs in which serine 5 is phosphorylated but that this phosphorylation shifts to serine 2 as transcription progresses TFIIH phosphorylates serine 5 and CTDK-1 (in yeast) phosphorylates serine 2 while cdk-9 phosphorylates serine 2 in higher eukaryotes 11-58

59 CTD phosphorylation patterns dictate which factors associate with RNAPII. RNAPII (gray oval) is depicted at four positions along a gene, and at each position its CTD (wavy line) is a different color to indicate different phosphorylation states: Gray indicates nonphosphorylated repeats; green indicates Ser5P repeats; red indicates Ser2,5P repeats (doubly phosphorylated); and blue indicates Ser2P repeats. Phatnani H P, and Greenleaf A L Genes Dev. 2006;20: Copyright 2006, Cold Spring Harbor Laboratory Press

60 Role of TFIIE and TFIIH TFIIE and TFIIH are not essential for the formation of an open promoter complex or for elongation Required for promoter clearance TFIIH has DNA helicase activity that is essential for transcription, presumably because it causes full melting of the DNA at the promoter and thereby facilitates promoter clearance 11-60

61 TFIIE Yeast 66kD and 43kD Humans 56kD and 34kD Required for in vitro transcription activity

62 TFIIH Yeast 9 subunits Humans 8 subunits Contains a kinase activity (cdk7) that can phosphorylate the CTD (serine 5) of the largest subunit of RNA polymerase II This phosphorylation is required for the enzyme to elongate transcription

63 Participation of General Transcription Factors in Initiation TFIID with TFIIB, TFIIF and RNA polymerase II form a minimal initiation complex at the initiator Addition of TFIIH, TFIIE and ATP allow DNA melting at the initiator region and partial phosphorylation of the CTD of largest RNA polymerase subunit These events allow production of abortive transcripts as the transcription stalls at about

64 Expansion of the Transcription Bubble Energy is provided by ATP DNA helicase of TFIIH causes unwinding of the DNA Expansion of the transcription bubble releases the stalled polymerase Polymerase is now able to clear the promoter 11-64

65 Transcription Factors in Elongation Elongation complex continues elongating the RNA when: Polymerase CTD is further phosphorylated by TEFb NTPs are continuously available TBP and TFIIB remain at the promoter TFIIE and TFIIH are not needed for elongation and dissociate from the elongation complex 11-65

66 Preinitiation complex.

67 Model for the participation of GTFs in initiation, promoter clearance, and elongation 11-67

68 Eukaryotic Control of Transcription Eukaryotes control transcription primarily at the initiation step There is also some control exerted during elongation, which can involve overcoming transcription pausing or transcription arrest RNA polymerases do not transcribe at a steady rate as they pause, sometimes for a long time, before resuming transcription Tend to pause at pause sites or DNA sequences that destabilize the DNA-RNA hybrid and cause the polymerase to backtrack 11-68

69 Promoter Proximal Pausing A sizable fraction of genes contain specific pause sites lying 20-50bp downstream of the transcription start site Two protein factors are known to help stabilize RNA polymerase II in the paused state - DRB sensitivity inducing factor (DSIF) and negative elongation factor (NELF) The signal to leave the paused state is delivered by the positive elongation factor-b (P-TEFb), which is a protein kinase that can phosphorylate polymerase II, DSIF, and NELF 11-69

70 TFIIS Stimulates Proofreading of Transcripts TFIIS stimulates proofreading, the correction of misincorporated nucleotides, likely by stimulating RNase activity of the RNA polymerase This would allow polymerase to cleave off a misincorporated nucleotide and replace it with a correct one 11-70

71 IIS

72 IIS

Eukaryotic & Prokaryotic Transcription. RNA polymerases

Eukaryotic & Prokaryotic Transcription RNA polymerases RNA Polymerases A. E. coli RNA polymerase 1. core enzyme = ββ'(α)2 has catalytic activity but cannot recognize start site of transcription ~500,000