Section M Transcription in Eukaryotes

1. Molecular Biology Course Section MTranscription in Eukaryotes

2. Section MTranscription in Eukaryotes • Topics • M1. The three RNA Polymerases: characterization and function • M2. RNA Pol I genes: the ribosomal repeat • M3. RNA Pol III genes: 5S and tRNA transcription • M4. RNA Pol II genes: promoters and enhancers • M5. General transcription factors and RNA Pol II initiation

3. Section MTranscription in Eukaryotes M1. The three RNA Polymerases: characterization and function • Eukaryotic RNA polymerases • RNA polymerase subunits • Eukaryotic RNA polymerase activities • The CTD of RNA Pol II

4. Section MTranscription in Eukaryotes M1-1: Eukaryotic RNA polymerases

5. Section M Transcription in Eukaryotes Main Features of eukaryotic transcription • The mechanism of eukaryotic transcription is similar to that in prokaryotes. • A lot more proteins are associated with the eukaryotic transcription machinery, which results in the much more complicated transcription.

6. Section M Transcription in Eukaryotes • 3.Three eukaryotic polymerases transcribe different sets of genes. The activities of these polymerases are distinguished by their sensitivities to the fungal toxin a-amanitin(鹅膏菌素). • 4. In addition, eukaryotic cells contain additional RNA Pols in mitochondria and chloraplasts.

7. Three eukaryotic polymerases

8. Section MTranscription in Eukaryotes M1-2: RNA polymerase subunits

9. Each eukaryotic polymerase contains 12 or more subunits. • the two largest subunits are similar to each other and to the b’ and b subunits of E. coli RNA Pol. • There is one other subunit in all three RNA Pol homologous to a subunit of E. coli RNA Pol. • Five additional subunits are common to all three polymerases. • Each RNA Pol contain additional four or seven specific subunit.

11. Section MTranscription in Eukaryotes M1-3: RNA polymerase activities • Transcription mechanism is similar to that of E. coli polymerase (How?) • Different from bacterial polymerasae, they require accessory factors for DNA binding.

12. Section MTranscription in Eukaryotes M1-4: The CTD of RNA pol II

13. The C-terminus of RNA Pol II contains a stretch of seven amino acids that is repeated 52 times in mouse enzyme and 26 times in yeast. • The heptapeptide sequenc is: Tyr-Ser-Pro-Thr-Ser-Pro-Ser • This repeated sequence is known as carboxyl terminal domain (CTD) • The CTD sequence may be phosphorylated at the serines and some tyrosines

14. 5. The CTD is unphosphorylated at transcription initiation, and phosphorylation occurs during transcription elongation as the RNA Pol II leaves the promoter (In vitro results). 6. Because it transcribes all eukaryotic protein-coding gene, RNA Pol II is the most important RNA polymerase for the study of differential gene expression. The CTD is an important target for differential activation of transcription elongation.

15. Section MTranscription in Eukaryotes M2. RNA Pol I genes:the ribosomal repeats

16. Section M Transcription in Eukaryotes 1-2: Structure of the rRNA genes 1. Ribosomal RNA genes 2. Role of the necleolus 3-6:RNA Pol I promoters & binding factors 3. RNA Pol I promoters 4. Upstream binding factor (UBF) 5. Selectivity factor 1 6. TBP and TAFIs 7: Other rRNA genes

17. Section MTranscription in Eukaryotes M2-1&2: Structure of the rRNA genes 1. Ribosomal RNA genes 2. Role of the necleolus

18. Ribosomal RNA Genes & nucleolus • A copy of 18S, 5.8S and 28S rRNA genes is organized as a single transcription unit in eukayotes. A 45S rRNA transcript (~13 000 nt long) is produced during transcription, which is then processed into 18S, 5.8S and 28S rRNA. • Pre-rRNA transcription units are arranged in clusters in the genome as long tandem arrays separated by nontranscribed spacer squences.

19. A single transcription unit Tandem array

20. Continuous transcription of multiple copies of rRNA genes by RNA Pol I is essential to produce sufficient rRNAs which are packaged into ribosomes. • The arrays of rRNA genes (rRNA cluster) loop together to form the nucleolus and are known as nucleolar organizer regions. • During active rRNA synthesis, the pre-rRNA transcripts are packaged along the rRNA genes, visualizing in the electronic microscope as “Christmas tree structures”.

21. Christmas Tree Structures

22. Section MTranscription in Eukaryotes M2-3~6: RNA Pol I promoters & binding factors 3. RNA Pol I promoters 4. Upstream binding factor (UBF) 5. Selectivity factor 1 6. TBP and TAFIs

23. RNA Pol I promoters • Generally consists of a bipartitesequence in the region preceding the start site, including core element and the upstream control elements (UCE). • RNA Pol I promoters in human cells are best characterized. • Core element: -45 to +20, sufficient for transcription initiatiation. • UCE: -180 to -107, to increase the transcription efficiency. • Both regions are rich in G:C, with ~85% identity.

24. RNA Pol I promoters in human cells Your text shows RNA Pol I promoter from another organism

25. Section M Transcription in Eukaryotes Two ancillary factors (UBF & SL1) of RNA Pol I & their roles in transcription initiation

26. Upstream binding factor(UBF) • A specific DNA-binding protein that binds to UCE, as well as a different site in the upstream of the core element, causing the DNA to loop between the two sites. (two binding sites have no obvious similarity) • UBF is essential for high level of transcription, and low level of expression occurs in its absence.

27. Selectivity factor 1(SL1) • Does not bind to promoters by itself • Binds to and stabilizes the UBF-DNA complex. • Interacts with the free downstream part of the core element. • Recruit RNA Pol I to bind and to initiate the transcription.

28. Subunits of SL1 • SL1 consists of 4 proteins. • TBP (TATA-binding protein): a factor also required for initiation by RNA Pol II and III. A critical general factor in eukaryotic transcription that ensures RNA Pol to be properly localized at the startpoint. • Other three subunits are referred to as TBP-associated factors (TAFIs) that are specific for RNA Pol I transcription.

29. The initiation complex assembles in three stages

30. TAFIs UBF RNA Pol I TBP TAFIs UBF TAFIs The initiation complex proposed in your text book It is not known which representation one is more accurate.

31. Other rRNA genes (simple) In a simple eukaryote, Acanthamoeba, the rRNA genes have only one control element (promoter) around 12-72 bp upstream from the transcription start site. Simple initiation: TIF (homolog of SL-1) binds to the promoter  RNA Pol I bind TIF remains bound and the RNA Pol I is released for elongation.

32. Section MTranscription in Eukaryotes M3. RNA Pol III genes: 5S and tRNA transcription • RNA polymerase III • tRNA genes • 5S rRNA genes • Alternative RNA Pol III promoters • RNA Pol III termination

33. Section MTranscription in Eukaryotes M3-1. RNA Pol III • Contains at least 16 or more subunits • Is located in nucloplasm • Synthesizes the precursors of 5S rRNA, the tRNAs and other small nuclear and cytosolic RNAs

34. Promoters for RNA polymerase III May consist of bipartite sequences downstream of the startpoint, with boxA separated from either boxC or boxB. Or they may consist of separated sequences upstream of the startpoint (Oct, PSE, TATA).

36. Section MTranscription in Eukaryotes M3-2. tRNA genes • The initial transcripts of tRNA genes need to be processed to produce the mature tRNA. • The transcription control regions of tRNA lies after the start site within the transcribed region. The two highly conserved control sequences are called A box(5’-TGGCNNAGTGG) and B box(5’-GGTTCGANNCC).

37. A box and B box also encode important sequences in the tRNA itself, the D-loop and TC-loop. • Therefore, the highly conserved sequence in tRNAs are also highly conserved promoter DNA sequences. • 3. Two complex DNA-binding factors required for tRNA transcription initiation: • TFIIIC---binds to both the A and B boxes, an assembly factor for positioning TFIIIB.

38. TFIIIB: (1)binds 50 bp upstream from the A box, but has no sequence specificity and the binding position is determined by the DNA bound TFIIIC. (2)consists of three subunits, one of which is TBP, the general initiation factor; the second is called BRF (TFIIB-related factor); and the third is called B”.

39. TFIIIC: A and B boxes binding and a assembly factor to position TFIIIB TFIIIB: DNA binding and RNA Pol III recruiting

40. Section MTranscription in Eukaryotes M3-3. 5S rRNA genes • Tandemly arranged in a gene cluster. (In human, there is a single cluster of around 2000 genes.) • Transcription control regions (promoters) are organized similar to those of tRNA, except that C box is in place of B box. C box: +81-99 bp; A box: +50-65

41. 3. Transcription factors:(1) The C box acts as the binding site for TFIIIA. (2) TFIIIA acts as an assembly factor which allows TFIIIC to interact with the 5S rRNA promoter. (3) The A box may also stabilize TFIIIC binding. (4) TFIIIC is then bound to DNA site near +1. (5) TFIIIB and TFIIIC interact to recruit RNA Pol III to initiate transcription.

42. TFIIIA TFIIIC TFIIIB Pol III

43. Section MTranscription in Eukaryotes M3-4. Alternative RNA Pol III promoters • Many RNA Pol III genes also rely on upstream sequences for regulation of their transcription • e.g. U6 snRNA and Epstein-Barr virus • Use only regulatory genes upstream from their transcription start sites.

44. U6 snRNA • The coding region contains a characteristic A box thatis not required for transcription. • The upstream sequence contains sequences typical of RNA Pol II promoters, including a TATA box at bases –30 to –23. • Shares several other transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II • Suggestion: common transcription factors can regulate both RNA Pol II and Pol III genes

45. Section MTranscription in Eukaryotes M3-5. RNA Pol III termination The RNA polymerase can terminate transcription without accessory factors. A cluster of A residue is often sufficient for termination. Xenopus borealis terminator: 5’-GCAAAAGC-3’

46. Section MTranscription in Eukaryotes M4. RNA Pol II genes: promoters and enhancers • RNA Pol II • Cis-acting elements • Promoters • Upstream regulatory elements • Enhancers

47. Section MTranscription in Eukaryotes M4-1. RNA Pol II • located in nucleoplasm • catalyzing the synthesis of the mRNA precursors for all protein-coding genes. • RNA Pol Ⅱ-transcribed pre-mRNAs are processed through cap addition, poly(A) tail addition and splicing.

48. Section MTranscription in Eukaryotes M4-2. Promoters • Most promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription. It has a 7 bp consensus sequence 5’-TATA(A/T)A(A/T)-3’. • TBP binds to TATA box that includes an additional downstream bp.

49. TATA box acts in a similar way to an E. coli promoter –10 sequence to position the RNA Pol II for correct transcription initiation. The spacing but not the sequence between the TATA box and the start site is important. Transcription starts with an adenine ~50% of the time.

50. Some eukaryotic genes contain an initiator element instead of a TATA box. The initiator element is located around the transcription start site. Other genes have neither a TATA box nor an initiator element, and usually are transcribed at very low rates.