1. Section M Transcription in Eukaryotes
Molecular Biology Course
Section M Transcription in Eukaryotes

2. Topics M1. The three RNA Polymerases: characterization and function
Section M Transcription 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. M1. The three RNA Polymerases: characterization and function
Section M Transcription 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. Eukaryotic RNA polymerases
Section M Transcription in Eukaryotes
M1-1:
Eukaryotic RNA polymerases

5. eukaryotic transcription
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
Type
Location
Substrate
a-amanitin
RNA Pol I
Nucleoli
Most rRNAs gene
Insensitive
RNA Pol II
Nucleo-plasm
All protein-coding genes and some snRNA genes
Very sensitive
RNA Pol III
tRNAs, 5S rRNA,
U6 snRNA and other small RNAs
Moderately sensitive

8. RNA polymerase subunits
Section M Transcription 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. RNA polymerase activities
Section M Transcription 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 M Transcription in Eukaryotes
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. M2. RNA Pol I genes: the ribosomal repeats
Section M Transcription in Eukaryotes
M2. RNA Pol I genes: the ribosomal repeats

16. 1-2: Structure of the rRNA genes
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. M2-1&2: Structure of the rRNA genes
Section M Transcription 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 (~ 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. M2-3~6: RNA Pol I promoters & binding factors 3. RNA Pol I promoters
Section M Transcription 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 bipartite sequence 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: 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. The initiation complex
proposed in your text book
UBF
RNA
Pol I
TBP
TAFIs
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 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. 5S and tRNA transcription
Section M Transcription 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. M3-1. RNA Pol III Contains at least 16 or more subunits
Section M Transcription 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 M Transcription 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 M Transcription 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: bp; A box:

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. M3-4. Alternative RNA Pol III promoters
Section M Transcription 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 that is 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. M3-5. RNA Pol III termination
Section M Transcription 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. M4. RNA Pol II genes: promoters and enhancers
Section M Transcription in Eukaryotes
M4. RNA Pol II genes: promoters and enhancers
RNA Pol II
Cis-acting elements
Promoters
Upstream regulatory elements
Enhancers

47. M4-1. RNA Pol II located in nucleoplasm
Section M Transcription 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 M Transcription in Eukaryotes
M4-2. Promoters
Most promoters contain a sequence called the TATA box around 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
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.

51. M4-3. Upstream regulatory elements
Section M Transcription in Eukaryotes
M4-3. Upstream regulatory elements

52. The basal elements (the TATA box and initiator elements) : primarily determine the location of the startpoint, and sponsor initiation only at a rather low level.
Upstream regulatory elements (URE) such as the SP1 box and CCAAT boxes, greatly increase the frequency of initiation. URE is located within bp from the promoter, and plays an important role in ensuring efficient transcription.

53. Section M Transcription in Eukaryotes
M4-4. Enhancers
Sequence elements which can activate transcription from thousands of base pairs upstream or downstream.

54. General characteristics of Enhancers
Exert strong activation of transcription of a linked gene from the correct start site.
activate transcription when placed in either orientation with respect to linked genes
Able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start site.
Exert preferential stimulation of the closets of two tandem promoters

55. M5. General transcription factors and RNA PolⅡ initiation
Section M Transcription in Eukaryotes
M5. General transcription factors and RNA PolⅡ initiation
RNA Pol II basal transcription factors
TFIID (TBP)
3. TFIIA
4. TFIIB and RNA Pol binding
5. Factors binding after RNA Pol.
6. CTD phosphorylation by TFIIH
7. The initiator complex

56. 1. TFIID:
Multiprotein Complex, including TBP, other proteins are known as TAFIIs.
TBP is the only protein binds to TATA box

57. TBP:
1. a general transcription factor bound to DNA at the TATA box.
2. a general transcription required by all 3 RNA pol.

58. TBP DNA TBP: 3. Has a saddle structure with an overall dyad symmetry.
Outer surface (with ?)
TBP
DNA
Inner surface (with ?)

59. TBP
45o Kink
TBP causes DNA bending

60. 2. TFIIA binds to TFIID stabilizes TFIID-DNA complex
contains at least 3 subunits

61. 3. TFIIB & RNA Pol binding
binds to TFIID
Binds to RNA Pol with TFIIF

62. 4-1 TFIIE binding
Necessary for transcription

63. 4-2 TFIIJ, TFIIH binding
Necessary for transcription

64. 5. phosphorylation of the polymerase CTD
by TFIIH
Formation of a processive RNA polymerase complex and allows the RNA Pol to leave the promoter region.

65. The initiator transcription complex
For TATA-box lacking RNA Pol II promoters, TBP is recruited to the initiator element 0verlapping the start site by some DNA-binding proteins, TBP then recruit the other transcription factors and polymerase similar to TATA box gene transcription.

66. Thanks