Section M: Transcription in EukaryotesYang Xu, College of Life Sciences 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

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences M1 The three RNA polmerases: Characterization and function Eukaryotic RNA polymerases Eukaryotic RNA polymerases activities RNA polymerase subunit

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences Eukaryotic RNA polymerases Classification: Three eukaryotic polymerases transcribe different sets of genes. Their activities are distinguished by their different sensitivities to the fungal toxin  -amanitin: RNA pol I:  transcribes most rRNA genes.  It is located in the nucleoli and is  insensitive to  -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  -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  -amanitin. In addition to these nuclear enzymes, eukaryotic cells contain additional RNA pol in mitochondria and chloroplasts.

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences Eukaryotic RNA polymerases Transcription for Location in Sensitivity to  -amanitin RNA pol I most of rRNA genes nucleoliinsensitive ( － ) RNA pol II mRNA and some snRNA nucleoplasmvery sensitive (+ +) RNA pol III tRNA and 5S rRNA nucleoplasmmoderately sensitive (+)

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences Activities of three RNA pol. Transcription in Eukaryotes Transcripition of rRNA gene Transcripition of tRNA and 5S rRNA gene Transcripition of mRNA and snRNA genes RNA Pol I RNA Pol IIRNA Pol III mRNA

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

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences M2 RNA pol. I genes: The ribosomal repeat Ribosomal RNA genes Promoter of RNA pol. I Upstream binding factor Selectivity factor 1 TBP and TAF I s Other rRNA genes

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences 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 nt long. 18S28S 5.8S Single transcr. U Tandem array 5‘3‘ Untranscribed spacer DNA This transcript is cleaved to give each copy of the 28S RNA (5000 nt), 18S (2000 nt) and 5.8S (160 nt) rRNAs.

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences 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 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. +1 Pre-rRNA gene -100 Core element UCE bp

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences UBF UBF-1 + UCE UBF-2 + Part of core promoter Two UBF interaction causing DNA to form a loop 3 TAF 1 TBP SL1 Upstream binding factor (UBF) UCE core promoter UBF

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

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences 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 TAF I s 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); TAF I s: 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 TAF I s. 3 TAF 1 TBP

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences 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

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences 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.

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences 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 ) A C C anticodon loop 5’ T  C-loop acceptor arm D loop V loop A boxB box

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences TFIIIC Fig. 1. Initiation of transcription at a eukaryotic tRNA promoter A boxB box TFIIIC B” TBP BRF RNA pol III B” TBP BRF TFIIIB RNA pol III

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences 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. B” TBP BRF TFIIIB TFIIIC

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences 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 bp downstream from the transcription start site. A box: A second sequence termed the A box around bases +50 to +65 is also important.

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences TFIIIC Fig. 2. Initiation of transcription at a eukaryotic 5S rRNA promoter TFIIIC RNA pol III B” TBP BRF TFIIIB TFIIIA A boxC box TFIIIA RNA pol III B” TBP BRF

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences Promoters of RNA pol. III 1. Eukaryotic tRNA promoter 2. Eukaryotic 5S rRNA promoter A boxB boxTATA box A boxC boxTATA box

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences 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.

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences M4 RNA pol II genes: Promoters and enhancers RNA polymerase II Promoters of RNA pol. II Upstream regulatory elements (URE) Enhancers

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences 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.

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 (37) (37) 1-4Kb C +1A GC island CAAC box TATA box Cap Enhancer UPE core promoter Promoter (basic factor) URE

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences 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 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.

Example: SV40 Enhancer (-179~ -250)

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

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences 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

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences RNA Pol II CTD F RNA Pol II F CTD 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. A TBP TAF II s A TFIIA B B TFIIB E E H H J J mRNA TBP TAF II s TFIID RNA Pol II CTD F TATA box

TIC: (Transcriptional Initiation Complex) Pre-TIC = Promoter + TFIID + TFIIA 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 TFIID: A sort of protein complex (TBP & > 8 TAF) TFIID and TFIIA TFIIA: Binds to TFII D Enhances TFII D binding to TATA box Stabilizing the DNA-TFII D complex TATA +1 Winds minor groove TFII A TFII D pre-TIC 1×3

TFIIB and 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) Basic TIC = Pre-TIC + TFIIB + [ RNA pol. II + TFIIF ] TFII F RNApol II TFII B Basic TIC 2×3

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 Factors binding after RNA polymerase TFIIJ: remains to be fully characterized. Complete TIC = Basic TIC + TFIIH + TFIIE + TFIIJ complete TIC EHEH J 3×3

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences TATA +1 Winds minor groove TFII A TFII D Pre -TIC TFII F RNApol II TFII B Basic TIC conclusion TIC: Transcriptional Initiation Complex

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences mRNA Promoter clearance Transcription starting complete TIC EHEH J E H B D A J 9－6＝39－6＝3

--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; The CTD of RNA pol. II Yeast 26X Drosophila 44X repeat unit of 7 aa / in CTD Rat & Human 52X

II O (240kd) II A (220kd) II B (180kd) With all Without allBasic form RNApol easy to leaves Promoter for elongation --CTD-end construction: 7aa repeats & high frequency phosphorylation Tyr—Ser P —Pro—Thr P —Ser P —Pro—Ser P

Section M: Transcription in EukaryotesYang Xu, College of Life Sciences That’s all for Section M