GEN409Presentation 5

GEN409Presentation 5 - Lecture...

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Unformatted text preview: Lecture 18 " " " "Chapter 20 Lewin’s Genes X! Eukaryotic Initiation of RNA Transcription! 1! How does sigma factor carry out this reaction? ! Look at transcription in E. coli:! "a) transcription in E. coli starts at a purine (> 90% of the cases). Site = +1! "b) 10 bases upstream from start site: At position -10! Note: there is no base assigned to 0 and the startpoint is +1. ! "CONSENUS SEQUENCE: -10 element: Pribnow Box! " " " "T A T A A T ! " " " " " "% of time that you will find these bases:! "T (80%) A (95%) T (45%) A (60%) A(50%) T (96%)! "c) At position -35! " " " "T T G A C A! " " " " Individual promoters usually differ from the consensus at one or more positions.! " "% of time that you will find these bases:! "T (82%) T (84%) G (78%) A (65%) C (54%) A (45%)! "d) distance between the -10 and -35 elements: = 16 base pairs in 90% of the time.! " "Thus, this spacing appears important:! " " " Start site is +1 "2 turns away on the double helix! "on the same side of the helix! "RNA polymerase comes in and binds to the same face of the helix.! 2 ! Chromatin must be opened before RNA polymerase can bind the promoter.! Figure 20.1! A typical gene transcribed by RNA polymerase II has a promoter that extends upstream from the site where transcription is initiated.! ! Cis elements ! recognized by transcription factors that are responsible for initiating transcription! !promoters are CIS elements – "usually 5’ of gene! " " "some cases, internal (for some RNA polymerase I genes)! 3! Initiation of RNA Transcription! Helpful definitions! Chromatin must be opened before RNA polymerase can bind the promoter.! •!basal transcription factors – Transcription factors required by RNA polymerase II to form the initiation complex at all RNA polymerase II promoters.! Factors are identified as TFIIX, where X is a letter.! •!core promoter – The shortest sequence at which an RNA polymerase can initiate transcription (typically at a much lower level than that displayed by a promoter containing additional elements).! For RNA polymerase II it is the minimal sequence at which the basal transcription apparatus can assemble, and it includes three sequence elements: the Inr, the TATA box, and the DPE.! It is typically ~40 bp long.! •!enhancer – A cis-acting sequence that increases the utilization of (most) eukaryotic promoters, and can function in either orientation and in any location (upstream or downstream) relative to the promoter.! •!silencer – A short sequence of DNA that can inactivate expression of a gene in its vicinity. ! •!housekeeping genes – Genes that are (theoretically) expressed in all cells because they provide basic functions needed for sustenance of all cell types.! •!coactivator – Factors required for transcription that do not bind DNA, but are required for (DNAbinding) activators to interact with the basal transcription factors.! •!preinitiation complex – The assembly of transcription factors at the promoter before RNA polymerase binds in eukaryotic transcription.! 4! Transcription in Eukaryotes! What strategies have been found?! Which TRANS factors participate and promote transcription? ! 3 Categories – defined by the Polymerase used! "! "RNA Polymerase " " " "location " "" amantin ! "1) RNA polymerase I – ribosomal genes "nucleolus "insensitive! "2) RNA polymerase II – messenger RNA "nucleoplasm "sensitive! "nucleoplasm " " "species ! "specific! "3) RNA polymerase III – "tRNAs " " "5s RNAs " " Heteronuclear RNA! " "sn RNAs! spiceosome complex hnRNA! " "! "large multi protein – 500,000 kd! " complexes 8-14 subunits! 5! Eukaryotic RNA polymerases consist of many subunits! •!All eukaryotic RNA polymerases have ~12 subunits (& genes encoding them) ! "and are aggregates of >500 kD.! •!Some subunits are common to all three RNA polymerases.! •!The largest subunit in RNA polymerase II has a CTD (carboxy-terminal domain) ! consisting of multiple repeats of a ! consensus sequence of 7 amino acids.! ~26 repeats in yeast ! and ~50 in mammals! !Genetic deletions that remove (typically) ! more than half of the CTD repeats are! lethal (in yeast).! CTD means c terminal domain. it undergoes phosphorilation. allows for synthesis of RNA •!CTD can be highly phosphorylated! on serine or threonine residues;! this is involved in the initiation ! of transcription! Figure 20.2! 6! Plasmid shuffle Assay:! is a procedure for screening of mutations, derived from a mutagenized plasmid, requiring the loss of a second plasmid to assay for the recessive mutations.! Yeast strain had several mutations:! "RNA polymerase II gene was mutant – lethal mutation! "Selection:! "Leu2 unable to grow on medium without Leucine, or a plasmid containing the wild-type Leu2 gene! "Ura3 Wild-type strains of yeast (or ura3 mutant strains containing a plasmid-borne URA3+ gene) are unable to grow on medium "containing the pyrimidine analog 5-fluoro-orotic acid 5’ FOA, whereas ura3- mutants grow normally. ! A series of CTD polymerase II deletions! were constructed, ! Thus RNA Polymerase II with 7 – 14 repeats! 2 plasmids:! Wild-type RNA polymerase II! Ura3 +! RNA polymerase II with deletions of CTD! Ura3 –! Leu + (allows growth on Leu - media)! Transform yeast strain with two plasmids ! Wild-type RNA polymerase II! Ura3 +! RNA polymerase II with 8 CTD repeats! Leu +! Grow on selective complete media minus Leucine, minus uridine = SC-Leu-Ura ! Result: yeast with both plasmids can grow! 8 ! Patch onto complete media for nonselective growth! Replica-plated onto SG-Leu +uridine plates ! Replica-plated onto SG-Leu +uridine plates supplemented with 5FOA! grown overnight! Select for growth yeast cells that have lost the Ura+ plasmid! 9 ! RNA polymerase II w ith 7 CTD repeats can not confer viability! RNA polymerase II with 8 CTD repeats grows! Thus:! ~26 repeats in wild type gene in yeast! 10 ! yeast strain z26 MATa his3A200 ura3-52 Leu2-3,112 rpblA187:: HIS3 GAL+(pRP112) ! FYI:! URA3 is a gene that encodes orotidine 5-phosphate decarboxylase (ODCase), an enzyme involved in the synthesis of pyrimidine ribonucleotides.! Loss of ODCase activity leads to a lack of cell growth unless uracil or uridine is added to the media (positive selection). In contrast, if 5-FOA is a dded to the media ODCase can convert into the toxic compound 5fluorouracil causing death (negative selection).! Since URA3 allows for both positive and negative selection, it has been developed as a genetic marker for DNA transformations and other genetic techniques in bacteria and many fungal species. It is one of the most important genetic markers in yeast genetic modification.! 11 ! PROMOTERS AND ENHANCERS ! are defined by Genetic and Biochemical experiments! "• in vivo and in vitro! "• Using forward and reverse Genetics ! •! Deletions of the DNA and Point mutations! in upstream sequences are useful in defining transcriptional control regions ! 12 ! For Example:! Mapping the 5S RNA promoter region using Deletion Analysis ! 5S RNAs are Synthesized by RNA polymerase III! 13 ! What strategies are used for the Initiation of Eukaryotic Transcription ?! In general:! "1) formation of a pre-initiation complex! essential for the polymerase to know where to bind "2) facilitates binding of RNA polymerase! "3) initiation of transcription! RNA polymerase I "transcribes ribosomal RNA genes (except 5S rRNAs)! rRNA genes! transcript includes sequences of both large and small rRNAs! rRNAs associate with ribosomal proteins in nucleolus! rRNA gene DNA: 0! precursor rRNA 5’ Mature RNAs 3’ 18S these rRNA genes are tandemly repeated 100 X $ $ repeats units 5.8S 28S # cleavage rRNAs makes ribosomes I think 28S !!! ! 5.8S base pairing (hydrogen bonds)! 14 Figure 20.3 *! predominately GC rich 2 transcription factors SL1physically positions DNA Tata binding protein=TBP never binds directly to the DNA RNA polymerase I! 15 ! Review: Human Cells Promoter bipartite DNA sequence: RNA polymerase I a) core promoter - surrounds the start site sufficient for transcription -45 to 20 UCE! 0! +1 not 0 Start is at 0 b) upstream element - 180 to -107 UCE = upstream control element " transcriptional efficiency. unusual base composition both core promoter and UCE high in GC content they are 85% identical Protein Factors: in addition to RNA polymerase I to initiate transcription Step A • UBF1 - binds to GC rich sequences in UCE (it enhances the ability of SL1 to bind) Step B • once UBF1 is bound to the DNA a 2nd factor SL1 binds to core promoter SL1 ! 4 proteins (one component is also required for poly II and poly III initiation = TBP) Step C • now RNA polymerase I binds 16 ! RNA polymerase III! ! Figure 20.4 17! RNA polymerase III! Internal type 2 promoter! distance between boxA and boxB varies, Transcription facter IIIc binds to boxA and B which allows TF IIIB bind to starting point. and then RNA poluermase III can bind. Figure 20.5 *! 18! RNA polymerase III! Internal type 1 promoter! Assembly factors TF III A binds to box A and boxC Recruits Positioning factor TFIIIB and then RNA polymerase III gets to starting point. TFIIIA and TFIIIC don't need to stay on the DNA for the poylermase to bind Figure 20.6 *! 19! SUMMARY:! 3 types of RNA polymerase III promoters! Internal promoters:! "RNA polymerase III + accessory factors ---! Initiation ! Type 1 promoters: ! "a) TFIIIA (zinc finger protein) binds to Box C! b) then TFIIIC binds [composed of at least 5 subunits]! c) then TFIIIB binds to a sequence surrounding the start point ! "[TBP + 11 other proteins (TAFs) = TFIIIB]! **** TFIIIB is the “true” initiation factor acts as a “positioning factor” and allows! RNA polymerase to localize properly to the promoter.! d) allows RNA poly III to bind at the start point. ! Type 2 promoters: ! "a) TFIIIC recognizes Box B and binds in the region of Box A and Box B. "b) then TFIIIB binds --! recruits RNA polymerase III! ! Upstream promoters:! Type 3 promoters:! "a) TATA Box is sufficient for initiation of transcription by RNA poly III, ! "b) while the OCT and PSE boxes increase promoter efficiency.! 20 ! RNA polymerase II! A minimal RNA polymerase II promoter has only two elements! Downstream Promoter ! Element at +28 to +32! RNA polymerase II as 3 cis elements. Core promoter and TATA box.-25 bp upstream of start point. it is relatively fixed in position. Flanked by GC, Initiator sequence-starts with A and flanked by T? I guess they overlap or something? IDK Don't need tata's to be a promoter. TATA box ~ 25 bp upstream of the ! "Inr (initiator) sequence [Py2CAPy5] between -3 and +5. ! Figure 20.7 21 ! *! RNA Polyermase II Transcription is highly regulated. TBP is tata binding protein. in polymerase II binds to TATA region. 50% or 10-20% don't have TATA's lots of promoters for Polymerase II •!TBP is a component of the positioning factor that is required for each type of RNA polymerase to bind its promoter.! •!The factor for RNA polymerase II is TFIID, which consists of TBP and ~14 TAFs, with a total mass ~800 kD.! Figure 20.8*! 22 ! TBP Is a Universal Factor ! •! TBP binds to the TATA box in the minor groove of DNA.! •! TBP forms a “saddle” around the DNA and bends it by ~80°. ! Composed of 2 subunits that bind to the TATA box and it binds in the minor grove of the DNA, unusal other Transcription Factors bind to the Major grove. The DNA is in gray with the two different strands and the TBP saddles the DNA. Puts force on the dna and bends it. Key:! Gray: double stranded DNA! TBP consists of two related (40%) conserved domains (light and dark blue), with a variable N-terminal domain.! Figure 20.9! TBP binds to the minor groove of DNA! 23! Figure 20.10! bending allows for the DNA to widen that then allows for the attachment of the TBP itself and we can see again the fact that this dimer binds to the dna and then bends to it which allows for other factors to bind and allow for the polymerase II to bind Co-crystal structure of TBP w ith DNA from -40 to the start point shows a bend at the TATA box that widens the narrow groove where TBP binds.! Attachment of TBP (blue) induces a bend in the DNA molecule.! The bend in the DNA (gray) opens up the minor groove, facilitating the attachment of TFIIB.! The Basal Apparatus Assembles at the Promoter! Gene promoters and chromatin structure! "Inactive gene, closed chromatin structure! "Potentially active gene, o pen chromatin: poised gene! " "assembly basal apparatus, needs a signal to start transcription! " "heat shock genes! when a gene is inactive and destined to be "Active gene, o pen chromatin conformation! completely inactive the chromatin is in a RNA polymerase II Promoters:! "core promoter: with start point! " " "Inr plus TATA or DPE ! closed conformation which is super packed. Transcription factors can't bind. In a heat shock gene which needs to be turned on quickly but not always. so it is in an open conformation so some factors are already on it but not the ones that make the polymerase bind to it. When genes are active the conformation is open with all the transcription factors and the polymerase With Additional elements: recognized by activators ! " " in addition we have other units that help "typically short sequences ~100 bases upstream of start! that are called activators and the have " " short sequences " " "binding influences initiation complex formation! "“mix and match” elements that contribute to promoter function! 25 ! The Basal Apparatus Assembles at the Promoter! RNA polymerase II Promoters:! "core promoter: TATA Step 1: " " " "TATA box is bound by the TFII D complex ! {! "TBP (TATA Binding Protein)! " " “saddles DNA”! "% TAF (TBP – associated factors)! " " " " " TATA core promoter bound by TFIID complex "! composed of TBF and TAF tatabinding associated factors protects -45 to -10 region. Important for the position of the polyerase. " "TFII D! " "#! "protects promoter in ! "-45 to –10 region in DNA! "foot printing experiments! "important for positioning RNA polymerase II! 26 ! Promoter region of DNA! DNA CIS ELEMENTS! "A) start point for transcription! "“in general” sequence = Py2 CA Py5! " " -3 +5! mRNA starts with an A flanked by pyrimidines! " "(thymidine cytosine)! "B) ~ 25 bp upstream !! " "TATA box! " "(almost identical to –10 box of prokaryotes)! " " (However, >50% of promoters are TATA less)! TATA box at start point, mRNA always starts with an A flanked by pyrimidines T or C typically 25 bp upstream of start point, once these factors are assembled polymerase can come in and allow for transcription to occur 27 ! LOOK OVER THIS PAGE Step 1: "TATA box is bound by the TFII D complex ! Step 2: joined by TFII A ! protects additional upstream sequences! Step 3: joined by TFII B ! binds downstream of TATA! "Now –45 to +10 is protected! Step 4:TFII F binds next! RAP 74 subunit – ATP dependent DNA helicase activity! to melt DNA at initiation start! RAP 38 subunit – interacts and binds RNA poly II! " " ! "(some sequence similarity with prokaryotic sigma factor) Step 5: RNA polymerase II binds! " Extends protected area to! "+15 template strand! "+ 20 nontemplate strand! "HUGE COMPLEX! Step 6: TFIIE ! binds further downstream +30 is protected.! Step 7: two additional factors are added:! "TFIIH [helicase activity: melts DNA]! "TFIIJ! Note: If a mutation occurs in the TATA sequence (centered at -30), transcription initiation ! occurs, but the start point varies. The TATA is crucial for correct positioning.! 28 ! The Basal Apparatus Assembles at the Promoter! TFIIB helps position RNA polymerase II w ith three domains of the protein interacting with RNA poly II:! o! N-terminal zinc ribbon contacts RNA poly II where the mRNA exits.! o! An “elongated" finger of TFIIB is inserted into the polymerase active center.! Figure 20.13! o! C-terminal domain interacts with RNA polymerase II and TFIID to orient the DNA.! 29 ! CTD is phosphorylated to remove factors and then polymerase can •!TFIIE and TFIIH are required to ! melt DNA to allow polymerase movement.! start making RNA Initiation is Followed by ! Promoter Clearance! •! Phosphorylation of the CTD (by TFIIH) ! may be required for elongation to begin.! •!TFIIH has multiple independent catalytic activities:! "ATPase! "helicase! XPD and XPB subunits! "kinase activity! CDK7 & cyclin H, phosphorylate serine amino acids on the RNA polymerase II Cterminal domain! •!TFIIH is also involved in DNA repair! •!At some genes RNA polymerase stutters, or makes only a short mRNA that is quickly degraded. ! •!RNA polymerase elongation requires the P-TEFb kinase! •!P-TEFb phosphorylates the CTD further! 30! For your information:! TBP Is a Universal Factor ! Universal and it also has also been found that when it is mutant it is associated with spinocerebellar ataxia 17 read more below. Figure 20.9! Key:! Gray: double stranded DNA! TBP consists of two related (40%) conserved domains (light and dark blue), with a variable N-terminal domain.! •!Another distinctive feature of TBP is a long string of glutamines in the N-terminus of the protein. This region modulates the DNA binding activity of the C-terminus, and modulation of DNA binding affects the rate of transcription complex formation and initiation of transcription. The number of CAG repeats encoding the polyglutamine tract is usually 32-39.! •!Two transcript variants encoding different isoforms have been found for this gene.! •!Mutations that expand the number of CAG repeats encoding this polyglutamine tract, and thus increase the length of the polyglutamine string, are associated with:! •!spinocerebellar ataxia 17, a neurodegenerative disorder classified as a polyglutamine disease.! 31! ...
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