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Section M Transcription in Eukaryotes

M1 The three RNA polmerases: Characterization and function M2 RNA pol. I genes: The ribosomal repeat M3 RNA pol. III genes: tRNA and 5S ribosomal repeat M4 RNA pol. II genes: Promoters and enhancers M5 General transcri. factors and pol. II initiation.

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Section M Transcription in Eukaryotes

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  1. M1 The three RNA polmerases: Characterization and functionM2 RNA pol. I genes: The ribosomal repeatM3 RNA pol. III genes: tRNA and 5S ribosomal repeatM4 RNA pol. II genes: Promoters and enhancersM5 General transcri. factors and pol. II initiation Section M Transcription in Eukaryotes Yang Xu, College of Life Sciences

  2. M1 The three RNA polmerases:Characterization and function • Eukaryotic RNA polymerases • Eukaryotic RNA polymerases activities • RNA polymerase subunit Yang Xu, College of Life Sciences

  3. Eukaryotic RNA polymerases Classification: Three eukaryotic polymerases transcribe different sets of genes. Their activities are distinguished by their different sensitivities to the fungal toxin a-amanitin: • RNA pol I:transcribes most rRNA genes.  It is located in the nucleoli and is  insensitive to a-amanitin. • RNA pol II: transcribes all protein-coding genes and some small nuclear RNA (snRNA) genes.  It is located in the nucleoplasm and is  very sensitive to a-amanitin. • RNA pol III:  transcribes the genes for tRNA, 5S rRNA, U6 snRNA and certain other small RNAs. It is located in the nucleoplasm and is  moderately sensitive to a-amanitin. In addition to these nuclear enzymes, eukaryotic cells contain additional RNA pol in mitochondria and chloroplasts. Yang Xu, College of Life Sciences

  4. Eukaryotic RNA polymerases Yang Xu, College of Life Sciences

  5. Transcription in Eukaryotes Transcripition of rRNA gene Transcripition of mRNA and snRNA genes Transcripition of tRNA and 5S rRNA gene RNA Pol I RNA Pol II RNA Pol III mRNA Activities of three RNA pol. Yang Xu, College of Life Sciences

  6. Including L, L’ subunit & 7-12 small subunits RNA polymerase subunit RNA pol. I, II, III L ~ β of prokaryoteRNA pol L’~ β’ of prokaryote RNA pol; L’ with 78% ± homologous among 3 RNA pol. (I, II, III)

  7. M2 RNA pol. I genes: The ribosomal repeat • Ribosomal RNA genes • Promoter of RNA pol. I • Upstream binding factor • Selectivity factor 1 • TBP and TAFIs • Other rRNA genes Yang Xu, College of Life Sciences

  8. Single transcr. U 18S 28S 5.8S Untranscribed spacer DNA Tandem array 5‘ 3‘ rRNA genes • RNA pol I is responsible for the continuous synthesis of rRNAs during inter-phase. • Human cells contain 5 clusters of around 40 copies of the rRNA gene situated in different chromosomes. • Each rRNA gene produces a 45S rRNA transcript which is about 13 000 nt long. • This transcript is cleaved to give each copy of the 28S RNA (5000 nt), 18S (2000 nt) and 5.8S (160 nt) rRNAs. Yang Xu, College of Life Sciences

  9. Yang Xu, College of Life Sciences

  10. UCE Core element 50-80 bp -31 +6 Pre-rRNA gene -100 +1 Promoter of RNA pol. I Pre-rRNA gene promoters: In mammalian, pre-rRNA gene promoters have a bipartite transcription control region: • Core element: includes the transcription  start site +1 and The -31 to +6 sequence, which is essential for transcription. • UCE: An additional element of around 50-80 bp named the upstream control element (UCE)  begins about 100 bp upstream from the start site (-100).  The UCE is responsible for an increase in transcription of around 10- to 100-fold compared with that from the core element alone. Yang Xu, College of Life Sciences

  11. UBF UBF UCE core promoter 3 TAF 1 TBP SL1 Upstream binding factor (UBF) • UBF • UBF-1 + UCE • UBF-2 + Part of • core promoter • Two UBF • interaction • causing DNA • to form a loop Yang Xu, College of Life Sciences

  12. RNApol I Pre-rRNA Allows RNA pol binding complex to initiate A low rate of basal transcription is seen in the absence of UBF, and this is greatly stimulated in the presence of UBF. Yang Xu, College of Life Sciences

  13. 3 TAF 1 TBP Selectivity factor 1 SL1: An additional factor, called selectivity factor, it is essential for RNA pol I transcription. • SL1 binds to and stabilizes the UBF-DNA complex and interacts with the free downstream part of the core element. • SL1 binding allows RNA pol I to bind to the complex and initiate transcription, and is essential for rRNA transcription. TBP and TAFIs • SL1 has now been shown to contain several subunits, including: • TBP (TATA-binding protein). TBP is required for initiation by all three eukaryotic RNA polymerases (see Ml, M3 and M5); • TAFIs: The other three subunits of SL1 are referred to as TBP-associated factors or TAFs, and those subunits required for RNA pol I transcription are referred to as TAFIs. Yang Xu, College of Life Sciences

  14. M3 RNA pol III genes: 5S and tRNA transcription • RNA polymerases III • tRNA genes • 5S rRNA genes • Promoters of RNA pol. III • RNA Pol III termination Yang Xu, College of Life Sciences

  15. RNA pol. III • Located in nucleoplasm • With at least 16 or more subunits • For the transcription for: • 4s pre-tRNA; • 5s pre-rRNA; • snRNA (small nuclear RNA); • Cytosolic RNA. Yang Xu, College of Life Sciences

  16. TC-loop A box B box A C C 5’ acceptor arm D loop V loop anticodon loop Transcription of tRNA genes • Cis-element including ( for pre-t RNA ) • two highly conserved regions --A box; TGGCNNAGTGG ( for coding D-loop of tRNA ) --B box; GGTTCGANNCC ( for coding TC-loop of tRNA ) Yang Xu, College of Life Sciences

  17. TFIIIB A box B box TFIIIC TFIIIC TBP TBP B” B” BRF BRF Fig. 1. Initiation of transcription at a eukaryotic tRNA promoter RNA pol III RNA pol III Yang Xu, College of Life Sciences

  18. TFIIIB TFIIIC TBP B” BRF Initiation of transcription at a euk. tRNA promoter TFIIIB binds 50 bp upstream from the A box. • TFIIIB consists of three subunits: • TBP, the general initiation factor required by all three RNA polymerases; • BRF (TFIIB-related factor), since this RNA pol II initiation factor has homology to TFIIB; • B" factor. • TFIIIB has no sequence specificity and therefore its binding site appears to be determined by the position of TFIIIC binding to the DNA. • TFIIIB allows RNA pol III to bind and initiate transcription. Once TFIIIB has bound, TFIIIC can be removed without affecting transcription. TFIIIC is therefore an assembly factor for the position of the initiation factor TFIIIB. Yang Xu, College of Life Sciences

  19. Transcription of 5S rRNA genes • 5S rRNA: RNA pol III transcribes the 5S rRNA of the large ribosomal subunit. This is the only rRNA subunit to be transcribed separately. • Tandem array: Like the other rRNA genes, the 5S rRNA genes are tandemly arranged in a gene cluster. In humans, there is a single cluster of around 2000 genes. • C box: The promoters of 5S rRNA genes contain an internal control region called the C box which is located 81-99 bp downstream from the transcription start site. • A box: A second sequence termed the A box around bases +50 to +65 is also important. Yang Xu, College of Life Sciences

  20. TFIIIB A box C box TFIIIC TFIIIC TBP TBP B” B” BRF BRF +81 +50 +99 +65 Fig. 2. Initiation of transcription at a eukaryotic 5S rRNA promoter TFIIIA RNA pol III TFIIIA RNA pol III Yang Xu, College of Life Sciences

  21. TATA box TATA box A box A box B box C box +1 +1 -30 -23 -30 -23 Promoters of RNA pol. III 1. Eukaryotic tRNA promoter 2. Eukaryotic 5S rRNA promoter Yang Xu, College of Life Sciences

  22. RNA pol. III termination • Termination of transcription by RNA pol III appears only to require polymerase recognition of a simple nucleotide sequence. • This consists of clusters of dA residues whose termination efficiency is affected by surrounding sequence. • For example, the sequence 5'-GCAAAAGC-3' is an efficient termination signal in the Xenopus somatic 5S rRNA gene. Yang Xu, College of Life Sciences

  23. M4 RNA pol II genes: Promoters and enhancers • RNA polymerase II • Promoters of RNA pol. II • Upstream regulatory elements (URE) • Enhancers Yang Xu, College of Life Sciences

  24. RNA polymerase II • RNA pol II is located in the nucleoplasm. • It is responsible for the transcription of all protein-coding genes (mRNA) and • some small nuclear RNA genes. • The pre-mRNAs must be processed after synthesis, including: • cap formation at the 5'-end of the RNA and • poly(A) addition at the 3'-end, as well as • removal of introns by splicing. Yang Xu, College of Life Sciences

  25. 1-4Kb -70 -30 -1C +1A GC island CAAC boxTATA boxCap -270 -170 Enhancer UPE core promoter Promoter (basic factor) URE Promoters of RNA pol. II TATA box / Hogness box / Goldberg-Hogness box (-30) Rich AT and rich GC flanked (function=–10 in E. coli) rich GC------T A T A ( A / T ) A ( A / T)------rich GC 82 97 93 85 63 (37) 83 50 (37)

  26. Upstream regulatory elements The low activity of basal promoters is greatly increased by the presence of other upstream elements of promoter. URE: These sequences which are often located within 100-200 bp upstream from the promoter (-170~-270),  and they are referred to as upstream regulatory elements (URE) and  play an important role in ensuring transcri-ption from the promoter. Two common examples are: • SP1box is found upstream of many genes both with and without TATA boxes, and • CCAAT box. Promoters may have one, both or multiple copies of these sequences. Yang Xu, College of Life Sciences

  27. Example: SV40 Enhancer (-179~ -250)

  28. Enhancer Promoter No for special gene only for special gene Enhancer Enhancervs. Promoter Enhance expression Basic expression Position not be fixed isolated region Bi-directional element Mono-directional element

  29. M5 General transcription factors and pol. II initiation • RNA Pol II basal transcription factors • TFIID and TBP • TFIIA, TFIIB and TFIIF • Factors binding after RNA polymerase • CTD phosphorylatiion by TFIIH • TIC: Transcriptional Initiation Complex Yang Xu, College of Life Sciences

  30. TATA box TFIID TAFIIs TAFIIs TBP TBP TFIIA A F B TFIIB RNA Pol II RNA Pol II mRNA CTD CTD RNA Pol II CTD F F RNA Pol II basal transcription factors • A series of nuclear transcription factors have been identified and purified. • These are required for the transcription initiation of RNA Pol II promoter. • These multisubunit factors are named TFIIA, TFIIB, etc. • They have been shown to assemble on basal promoters in a specific order. E H B A J H E J Yang Xu, College of Life Sciences

  31. TATA +1 pre-TIC TFII ATFII D Winds minorgroove TFIID and TFIIA 1×3 TIC: (Transcriptional Initiation Complex) Pre-TIC = Promoter + TFIID + TFIIA TFIID: A sort of protein complex (TBP & > 8TAF) • TBP: TATA-binding protein • Needed for RNApol I, II, III (SL1, TFIIIB, TFIID) • Very high conserved C-end domain of 180 aa • Binds with DNA in minor groove & winds it TFIIA: Binds to TFII D Enhances TFII D binding to TATA box Stabilizing the DNA-TFII D complex

  32. TFII F RNApol II TFII B Basic TIC TFIIB and TFIIF 2×3 Basic TIC = Pre-TIC + TFIIB + [ RNA pol. II + TFIIF ] TFIIB: Once TFIID has bound to DNA, TFIIB binds to TFIID and binds to TBP of TFIID and RNA pol. II This seems to be; TFIIB as a bridging factor allowing recruitment of RNApol II to TIC together with TFIIF TFIIF: Including two subunits of RAP30, RAP74 Binding and recruiting RNApol II to assemble TIC Promoting RNA elongation by its helicase (ATPase)

  33. complete TIC J E H Factors binding after RNA polymerase 3×3 Complete TIC = Basic TIC + TFIIH + TFIIE + TFIIJ TFIIH: Large complex made up of > 5 subunits Helicase activity (ATPase) & kinase activity Phosphorylation of CTD of RNApol II DNA repair TFIIE: Associate with TFIIH kinase TFIIJ: remains to be fully characterized.

  34. TATA +1 TFII A TFII D TFII F RNApol II TFII B TIC: Transcriptional Initiation Complex conclusion Pre -TIC Winds minor groove Basic TIC Yang Xu, College of Life Sciences

  35. J J E H E H B D A mRNA complete TIC Promoter clearance 9－6＝3 Transcription starting Yang Xu, College of Life Sciences

  36. Yeast 26X Drosophila 44X repeat unit of 7 aa / in CTD Rat & Human 52X The CTD of RNA pol. II --L’ max. subunit is 240 kd & has specific COOH-end named CTD ( Carboxyl Terminal Domain ) only in RNApol II --CTD is essential for viability of enzyme;

  37. --CTD-end construction: 7aa repeats & high frequency phosphorylation Tyr—SerP—Pro—ThrP—SerP—Pro—SerP IIO(240kd) IIA (220kd)IIB (180kd) With all Basic form Without all RNApol easy to leaves Promoter for elongation

  38. That’s all for Section M Yang Xu, College of Life Sciences

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