M1 The three RNA polymerases: characterization and function

1. Section M—Transcription in eukaryotes Molecular Biology • M1 The three RNA polymerases: characterization and function • M2 RNA Pol Ⅰ genes: the ribisomal 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

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

3. Molecular Biology 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 polyneraseⅡ(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. Molecular Biology There Are Many Functional Classes of RNA mRNA rRNA tRNA snRNA scRNA 7S RNA micro RNA

5. Molecular Biology RNA polymerase subunits • – 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.

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

7. Molecular Biology Eukaryotic RNA polymerase activities • 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.

8. Molecular Biology 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. Molecular Biology M2 RNA PolⅠ 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 18S 5.8S 28S Transcription 45S transcript 18S 5.8S 28S 5¢ 3¢ Cleavage (the light pink regions are degraded) 18S 5.8S 28S rRNA rRNA rRNA Molecular Biology Ribosomal RNA transcription units

11. Molecular Biology Role of the nucleolus 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.

12. Molecular Biology 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. Molecular Biology Upstream binding factor 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 Ⅰtranscription. 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. TAFI s（TBP-associated factors）：as the other subunits of SL1 and required for RNA polyⅠtranscription called TAFⅠs。 Schematic model for rRNA transcription initiation

14. Molecular Biology M3 RNA Pol ⅢGenes: 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. Molecular Biology 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 TFⅢB. Initiation of transcription at a eukaryotic tRNA promoter

16. Molecular Biology 5S rRNA gene transcription 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 TFⅢA relative to the tRNA transcription initiation. TFⅢA acts to bind to C box and stabilize the interaction between TFⅢC and 5S rRNA. Initiation of transcription at a eukaryotic 5S rRNA promoter

17. Molecular Biology Alternative RNA Pol Ⅲ promoters and RNA Pol Ⅲ termination 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.

18. Molecular Biology 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.