1. M1 The three RNA polymerases: characterization M1 The three RNA polymerases: characterization and function and function M2 RNA Pol Ⅰ genes: the ribisomal repeat M2 RNA Pol Ⅰ genes: the ribisomal repeat M3 RNA Pol Ⅲ genes: 5S and tRNA transcription M3 RNA Pol Ⅲ genes: 5S and tRNA transcription M4 RNA Pol Ⅱ genes: promoters and enhancers M4 RNA Pol Ⅱ genes: promoters and enhancers M5 General transcription factors and RNA Pol Ⅱ M5 General transcription factors and RNA Pol Ⅱ initiation initiation Section M—Transcription in eukaryotes

2. Transcription and its control are much more complex in eukaryotes. There are 3 RNA polymerases, each specific for a subset of RNAs. M1 The three RNA polymerases: characterization and function

3. Eukaryotic RNA polymerases The mechanism of eukaryotic transcription is similar to that in prokaryotes. However, the large number of polypeptides associated with the eukaryotic transcription machinery makes it far more complex. Three different RNA polymerase complexes are responsible for the transcription of different types of eukaryotic genes. The different RNA polymerases were identified by chromatographic purification of the enzymes and elution at different salt concentrations(Topic B4). Each RNA polymerase has a different sensitivity to the fungal toxin α-amanitin and this can be used to distinguish their activities. ● RNA polynerase Ⅰ (RNA pol Ⅰ ) transcribes most rRNA genes. It is located in the nucleoli and is insensitive to α-amanitin. ● RNA polynerase Ⅱ (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 α- amanitin. ●RNA polynerase Ⅲ (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 α-amanitin. In addition to these nuclear enzymes, eukaryotic cells contain additional polymerase in mitochondria and chloroplasts.

4. mRNA rRNA tRNA snRNA scRNA 7S RNA micro RNA There Are Many Functional Classes of RNA

5. – 500-700kDa, 12+ subunits, most of RNA pol II structures are determined. The genes encoding the two largest subunits of each RNA polymerase have homology to each other.  The largest subunits of each eukaryotic RNA polymerase is similar to the β’ subunit of the E. coli polymerase, and the second largest subunit is similar to the βsubunit which contains the actiove site of the E. coli enzyme. Two subunits which are common to RNA Pol I and RNA PolIII, and a further subunit which is specific to RNA Pol II, have homology to the E. coli RNA polymerase αsubunit. At least five other smaller subunits are common to the three different polymerases. Each polymerase also contains an additional four to seven subunits which are only present in one type. RNA polymerase subunits

6. Similar Structures of Bacterial (left) and Eukaryotic (right) RNA Polymerases

7. Like bacterial RNA polymerases, each of the eukaryotic enzymes catalyzes transcription in a 5’ to 3’ direction and synthesizes RNA complementary to the antisense template strand. The reaction requires the precursor nuckeotides ATP ， GTP ， CTP and UTP and does not requires a primer for transcription initiation. The purified eukaryotic RNA polymerases, unlike the purified bacterial enzymes, require the presence of additional initiation proteins before they are able to bind to promoters and initiate transcription. Eukaryotic RNA polymerase activities

8. The CTD of RNA Pol II CTD---C-terminal domain RNA Pol II RPB1 subunit has (CTD) with repeat (YSPTSPS)n, n=26-52, In vitro studies have shown that the CTD sequence may be phosphorylated at the serines and tyrosines. Phosphorylate /Unphosphorylated Unphosphorylated to initiate transcription Phosphorylated for elongation

9. M2 RNA Pol Ⅰ genes: the ribosomal repeat RNA polymerase Ⅰ ( RNA pol Ⅰ ) is responsible for the continuous synthesis of rRNA during interphase. Human cells contain five clusters of around 40 copie of rRNA gene situated on different chromosomes (see Fig.1 and Topic D4). Each rRNA gene produces a 45S rRNA transcript which is about 13000 nt long(see the Topic D4). This transcript is cleaved to give one copy each of the 28S RNA (5000 nt), 18S(2000nt) and 5.8S (160 nt) rRNA (see Topic O1). The continuous transcription of multiple gene copies of the RNAs is essential for sufficient production of the processed rRNAs which are packaged into ribosomes.

10. Promoter Transcription Cleavage (the light pink regions are degraded) 45S transcript 18S5.8S28S 53 18S5.8S28S 18S5.8S28S rRNA Ribosomal RNA transcription units

11. Each rRNA cluster is known as a nucleolar organizer region, since the nucleolus contains large loops of DNA correspondind to the gene clusters. After a cell emerges from mitosis, rRNA synthesis restarts and tiny nucleoli appear at the chromosomal locations of the rRNA genes. During active rRNA synthesis, the pre-rRNA transcripts are packed along the rRNA genes and may be visualized in the electron microscope as ‘Chrismas tree structures’. In these structures, the RNA transcripts are densely packed along the DNA and stick out perpendicularly from the DNA. Short transcripts can be seen at the start of the gene, which get longer until the end of the transcription unit, which is indicated by disappearance of the RNA transcripts. Role of the nucleolus

12. RNA Pol I promoters Mammalian pre-rRNA gene promoters have a bipartite transcription control region(Fig.2). The core element includes the transcription start site and encompasses bases -31 to +6. This sequence is essential for transcription. 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.

13. UBF ： A specific DNA-binding protein,called upstream binding factor, binds to the UCE SL1: An additional factor called selectivity factor 1, is essential for RNA Pol Ⅰ transcription. SL1 binds to and stabilizes the UBF-DNA complex and interacts with free downstream part of the core element. TBP ： One of the subunits of SL1, called TATA-binding protein, is required for initiation by all three eukaryotic RNA polymerases. TAF I s （ TBP-associated factors ） ： as the other subunits of SL1 and required for RNA poly Ⅰ transcription called TAF Ⅰ s 。 Upstream binding factor Schematic model for rRNA transcription initiation

14. M3 RNA Pol Ⅲ Genes: 5S and tRNA transcription RNA polymerase Ⅲ (RNA poly Ⅲ ) is a complex of at 16 defferent subunits. Like RNA Pol Ⅱ, it is located in the nucleoplasm. RNA polymerase Ⅲ synthesizes the precursors of 5S rRNA, the tRNAs and snRNA and cytosolic RNAs.

15. tRNA gene transcription Why are the highly conserved sequences within the tRNA also highly conserved promoter DNA sequences? A box ： 5’-TGGCNNAGTGG-3’ B box ： 5’-GGTTCGANNCC-3’ TFIIIB and TFIIIC are required for tRNA gene transcription. TFIIIB allows RNA Poly Ⅲ to bind and initiate transcription. TFIIIC is an assembly factor for the positioning of the initiation factor TF Ⅲ B. Initiation of transcription at a eukaryotic tRNA promoter

16. 5S rRNA gene transcription Initiation of transcription at a eukaryotic 5S rRNA promoter The promoters of the 5S rRNA genes contain C box and A box as internal control regions. 5S rRNA transcription initiation needs an additional assembly factor TF Ⅲ A relative to the tRNA transcription initiation. TF Ⅲ A acts to bind to C box and stabilize the interaction between TF Ⅲ C and 5S rRNA.

17. Many RNA Pol III genes also rely on upstream sequences for the regulation of their transcription. Some promoters such as the U6 small nuclear RNA (U6 snRNA ) and small RNA genes from the Epstein-Barr virus use only regulatory sequences upstream from their transcription start sites. The coding region of the U6 snRNA has a characteristic A box. However, this sequence is not required for transcription. The U6 snRNA upstream sequence contains sequence typical of RNA Pol II promoters, including a TATA box at bases -30 to -23. these promoters also share several other upstream transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II. These observations suggest that common transcription factors can regulate both RNA Pol II and RNA Pol III genes. Alternative RNA Pol Ⅲ promoters and RNA Pol Ⅲ termination

18. RNA Pol Ⅲ termination Termination of transcription by RNA Pol Ⅲ appears only to require polymerase recognition of a simple nucleotide sequence consisting of dA residues, whose termination efficiency is affected by surrounding sequence. Thus the sequence 5’-GCAAAAGC-3’ is an efficient termination signal in the Xenopus borealis somatic 5SrRNA gene.

19. M4 RNA Pol II genes: promoters and enhancers RNA Polymerase II (RNA Pol II) is located in the nucleoplasm. It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes. The pre-mRNAs must be processed after synthesis by 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.

20. TATA box Transcription Transcriptional start site DNA Coding-strand sequences: TATAAAA GC + CAAT boxes –100 –50–25 +1 Py 2 CAPy 5 Promoters TATA box: Many eukaryotic promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription. It has the 7 bp consensus sequence 5’-TATA （ A/T ） A （ A/T ） -3’ although it is now known that the protein which binds to the TATA box, TBP, binds to an 8 bp sequence that includes an additional downstream base pair, whose identity is not important. Initiator element: The initiator element is located around the transcription strt site. Many initiator elements have a C at -1 and A at +1.

21. Upstream regulatory elements These elements are found in many genes which vary widely in their levels of expression in different tissues. Two common examples are SP1 box, which is found upstream of many genes both with and without TATA boxes, and the CCAAT box. Promoters may have one, both or multiple copies of these sequences. These sequences which are often located within 100-200 bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter.

22. Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site. This kind of elements are called as enhancer. Classically, enhancers have the following general chracteristics: They exert strong activation of transcription of a ;linked gene from the correct start site. They activate transcription when placed in either orientation with respect to linked genes. They are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start site. They exert preferential stimulation of the closest of two tandem promoters. Enhancers

23. M5 General transcription factors and RNA Pol II initiation RNA Pol II basal transcription fators ： A serial of nuclear transcription factors have been identified, purified amd cloned. These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA ， TFIIB ， TFIIC ， TFIID. They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation. TFIID ： Inn promoters containing a TATA box, the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element. The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex. It seems that in mammalian cells, TBP binds to the TATA box and is then joined by at least eight 由 TBP 和 TAF IIs to form TFIID.

24. TBP TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation. TBP is a monomeric protein, with a highly conserved C- terminal domains of 180 residues and this conserved domain functions as well as the full- length protein in in vivo transcription.

25. TBP structure TBP has been shown to have saddle structure with an overall dyad symmetry, but two halves of the molecule are not identical. TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors. Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound. This results in a kink of about 45° between the first two and last two base pariss of the 8 bp TATA element.

26. TFIIA, TFIIB and RNA polymearse binding TFIIA: TFIIA binds to TFIID and enhances TFIID binding to the TATA box, stabilizing the TFIID-DNA complex. TFIIA is made up of at least three subunits. TFIIB and RNA polymearse binding: Once TFIID has bound to the DNA, another transcription factor, TFIIB, binds to TFIID. TFIIB can also bind to the RNA polymerase. This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator, TFIIF.

27. Factors binding after RNA polymerase After RNA polymerase binding, three other transcription factors, TFIIE, TFIIH, and TFIIJ, rapidly asociate with the compex. These proteins are necessary for transcription in vitro and associate with the complex in a defined order. TFIIH is a large compex which is made up of at least five subunits. TFIIJ remains to fully characterized.

28. CTD phosphorylation by TFIIH TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity. Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase. This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region. TFIIH therefore seems to have a very important function in control of transcriptiom elongation.

29. TFIID TFIIB TFIID TFIIF TATA box TFIIB binds to TFIID. TFIIB acts as a bridge to bind to RNA polymerase II/TFIIF. TFIIE and TFIIH bind to RNA polymerase II. RNA polymerase TFIIB TFIIA TFIIA binds to TFIID and enhances TFIID binding to TATA box. Once TFIID has bound to the DNA ， TFIIB binds to TFIID.

30. After RNA polymerase binding, three other transcription factors, TFIIE, TFIIH, and TFIIJ, rapidly asociate with the compex Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase. This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region. Transcription factors

31. The initiator transcription complex Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site. It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element. TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters.

32. Vmax = 50 nts / sec  About 1/10th DNA polymerase  Which needs to be fast:  Initiated at very few points  Only ~ 10 DNA pol molecules / cell  Each has to be very fast  ~3,000 molecules / cell  Transcription simultaneously at many points RNA pol. Speed

33. Error rate of ~ 10 -4 to 10 -5.  Much greater than DNA pol  Less than expected just from W.-C base-pairing  Suggests proof-reading Details of proof-reading not understood. RNA pol. Fidelity

34. What about the dsDNA sequence tells RNA polymerase where to start? Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer. Homework