Section M – Transcription in eukaryotes

M1 The three RNA polymerases: characterization and function Eukaryotic RNA polymerase, RNA polymerase subunits, Eukaryotic RNA polymerase activities, The CTD of RNA Pol ⅡEukaryotic RNA polymeraseRNA polymerase subunitsEukaryotic RNA polymerase activitiesThe CTD of RNA Pol Ⅱ M2 RNA Pol Ⅰ genes: the ribosomal repeat Ribosomal RNA genes, Role of the nucleolus, RNA Pol Ⅰ promoters, Upstream binding factor, Selectivity factor 1, TBP and TAF 1 s, Other rRNA genesRibosomal RNA genesRole of the nucleolusRNA Pol Ⅰ promoters Upstream binding factorSelectivity factor 1TBP and TAF 1 sOther rRNA genes 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 terminationRNA polymerase ⅢtRNA genes5s rRNA genesAlternative RNA Pol Ⅲ promotersRNA Pol Ⅲ termination M4 RNA Pol Ⅱ genes: promoters and enhancers RNA polymerase Ⅱ, Promoters, Upstream regulatory elements, EnhancersRNA polymerase ⅡPromotersUpstream regulatory elements Enhancers M5 General transcriptiion factors and RNA Pol Ⅱ initiation RNA Pol Ⅱ basal transcription factors, TF Ⅱ D, TBP, TF Ⅱ A, TF Ⅱ B and RNA polymerase binding, Factors binding after RNA polymerase, CTD phosphorylation by TF Ⅱ H, The initiator transcriptiom complexRNA Pol Ⅱ basal transcription factorsTF Ⅱ DTBPTF Ⅱ ATF Ⅱ B and RNA polymerase bindingFactors binding after RNA polymerase CTD phosphorylation by TF Ⅱ HThe initiator transcriptiom complexContents

M1 The three RNA polymerases: characterization and function — Eukaryotic RNA polymerase Three eukaryotic polymerases transcribe different set of genes. Their activities are distinguished by their sensitivities to the fungal toxin( 毒素 ) α-amanitin (α- 鹅膏 蕈碱 ). RNA polymerase I is located in the nucleoli. It’s responsible for the synthesis of the precursors of most rRNAs. RNA polymerase II is located in the nucleoplaam and is responsible for the synthesis mRNA precursors and some small nuclear RNAs. RNA polymerase III is located in the nucleoplasm.It’s responsible foe the synthesis of the precursors of 5s rRNA, tRNAs and other small nuclear and cytosolic( 细胞 溶质的 ) RNAs.

TypeLocationSubstrate  -amanitin RNA Pol INucleoliMost rRNAs geneInsensitive RNA Pol IINucleo- plasm All protein-coding genes and some snRNA genes Very sensitive RNA Pol IIINucleo- plasm tRNAs, 5S rRNA, U6 snRNA and other small RNAs Moderately sensitive Three eukaryotic polymerases

M1 The three RNA polymerases: characterization and function — RNA polymerase subunits –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.

M1 The three RNA polymerases: characterization and function — Eukaryotic 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.

M1 The three RNA polymerases: characterization and function — The CTD of RNA Pol Ⅱ 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

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.

M2 RNA Pol Ⅰ genes: the ribosomal repeat — Ribosomal RNA genes 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.

3.Continuous transcription of multiple copies of rRNA genes by RNA Pol I is essential to produce sufficient rRNAs which are packaged into ribosomes. 4.The arrays of rRNA genes (rRNA cluster) loop together to form the nucleolus and are known as nucleolar ( 核仁的 )organizer regions. 5.During active rRNA synthesis, the pre-rRNA transcripts are packaged along the rRNA genes, visualizing in the electronic microscope as “Christmas tree structures”.

M2 RNA Pol Ⅰ genes: the ribosomal repeat — Role of the nucleolus Pre-rRNA is synthesized by RNA polymerase I (RNA Pol I) in the nucleolus. The arrays of rRNA gene loop together to form the nucleolus and are know as nucleolar organizer regions.

A promoter is the site on the DNA to which an RNA polymerase molecule binds prior to initiating transcription. We should clearly understand what a promoter is before further study ? Let’s review the concept.

Antisense or template DNA strand : the strand from which it copies. Sense or coding strand: the other strand, to which it is identical RNA.

The nucleotide +1 in transcription: The nucleotide at which transcription is initiated as +1. The nucleotide -1 in transcription: The nucleotide that precedes nucleotide +1 is initiated as -1.

Upstream: Those portions of the DNA preceding the initiation site(or start point)(toward the 3` end of the template). Downstream: Those portions of the DNA succeeding the initiation site(or start point)(toward the 5` end of the template).

Prokaryotic promoter sequences -10 position ( called Pribnow box,TATAAT) -35 position (TTGACA) Be absolutely essential to start transcription in prokaryotes. Allows a very high transcription rate.

Then,we can know that the bacterial promoter almost always contains some version of the following elements:

Eukaryotic promoters are extremely diverse and are difficult to characterize. They typically lie upstream of the gene and can have regulatory elements several kilobases away from the transcriptional start site. Eukaryotic promoters (Fig.11.19)

Many eukaryotic promoters, but by no means all, contain a core promoter element, a TATA box (sequence 5`-TATAAA-3`), which in turn binds a TATA binding protein which assists in the formation of the RNA polymerase transcriptional complex.

The TATA box typically lies very close to the transcriptional start site (often within 50 bases). Eukaryotic promoter regulatory sequences typically bind proteins called general transcription factors(GTFs) which are involved in the formation of the transcriptional complex.

M2 RNA Pol Ⅰ genes: the ribosomal repeat — 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: -180 to -107, to increase the transcription efficiency. Both regions are rich in G:C, with ~85% identity.

M2 RNA Pol Ⅰ genes: the ribosomal repeat — Upstream binding factor 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

M2 RNA Pol Ⅰ genes: the ribosomal repeat — Selectivity factor 1 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.

M2 RNA Pol Ⅰ genes: the ribosomal repeat — TBP and TAF 1 s 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.

The initiation complex assembles in three stages

M2 RNA Pol Ⅰ genes: the ribosomal repeat — Other rRNA genes 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.

M3 RNA Pol Ⅲ genes: 5s and tRNA transcription — 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).

M3 RNA Pol Ⅲ genes: 5s and tRNA transcription — 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).

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.

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”.

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

M3 RNA Pol Ⅲ genes: 5s and tRNA transcription — 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:

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.

M3 RNA Pol Ⅲ genes: 5s and tRNA transcription — 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.

U6 snRNA 1.The coding region contains a characteristic A box that is not required for transcription. 2.The upstream sequence contains sequences typical of RNA Pol II promoters, including a TATA box at bases –30 to –23. 3.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

M3 RNA Pol Ⅲ genes: 5s and tRNA transcription — 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’

M4 RNA Pol Ⅱ genes: promoters and enhancers — RNA polymerase 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.

M4 RNA Pol Ⅱ genes: promoters and enhancers — 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.

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. 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.

M4 RNA Pol Ⅱ genes: promoters and enhancers — Upstream regulatory elements 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.

M4 RNA Pol Ⅱ genes: promoters and enhancers — Enhancers Enhancers Enhancers : Sequence elements which can activate transcription from thousands of base pairs upstream or downstream.

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

M5 General transcriptiion factors and RNA Pol Ⅱ initiation — RNA Pol Ⅱ basal transcription factors A complex series of basal transcription factors have been characterzed which bind to RNA Pol II promoters and together initiate transcription. These factors and their component subunits are still being identified. They were originally named TFIIA, TFIIB TFIIC.

M5 General transcriptiion factors and RNA Pol Ⅱ initiation — TF Ⅱ D Multiprotein Complex, including TBP, other proteins are known as TAFIIs. TBP is the only protein binds to TATA box

M5 General transcriptiion factors and RNA Pol Ⅱ initiation — TBP 1. a general transcription factor bound to DNA at the TATA box. 2. a general transcription required by all 3 RNA pol. 3. Has a saddle structure with an overall dyad symmetry. TBP DNA Outer surface (with ?) Inner surface (with ?)

M5 General transcriptiion factors and RNA Pol Ⅱ initiation — TF Ⅱ A TFIIA binds to TFIID stabilizes TFIID-DNA complex contains at least 3 subunits

M5 General transcriptiion factors and RNA Pol Ⅱ initiation — TF Ⅱ B and RNA polymerase binding TFIIB & RNA Pol binding binds to TFIID Binds to RNA Pol with TFIIF

M5 General transcriptiion factors and RNA Pol Ⅱ initiation — Factors binding after RNA polymerase After RNA polymerase binding, TFIIE, TFIIH and TFIIJ associate with the transcription complex in a defined binding sequence. Each of these proteins is required for transcription in vitro.

4-1 TFIIE binding Necessary for transcription

4-2 TFIIJ, TFIIH binding Necessary for transcription

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.

M5 General transcriptiion factors and RNA Pol Ⅱ initiation — CTD phosphorylation by TF Ⅱ H TFIIH phosphorylates( 使磷酸化 ) the carboxy-terminal domain (CTD ，羟基末端 结构域 ) of RNA Pol II. This results in formation of a processive polymerase complex.

M5 General transcriptiion factors and RNA Pol Ⅱ initiation — The initiator transcriptiom 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.

Multiple choice questions 1. Which one of the following statements about eukaryotic RNA polymerases I, II and III is false? A RNA Pol II is very sensitive toα-amanitin. B RNA Pol II is located in th~ nucleoplasm. C RNA Pol III transcribes th~ genes for tRNA. D eukaryotic cells contain other RNA polymerases in addition to RNA Pol I, RNA Pol II and RNA Pol III. E each RNA polymerase contains subunits with homology to subunits of the E. coli RNA polymerase as well as additional subunits ， which are unique to each polymerase. F the carboxyl end of RNA Pol II contains a short sequence of only seven amino acids which is called the carboxyl-terminal domain (CTD) and which may be phosphorylated. 2. Which two of the following statements about RNA Pol I genes are true? A RNA Pol I transcribes the genes for ribosomal RNAs. B human cells contain 40 clusters of five copies of the rRNA gene. C the 185, 5.85 and 285 rRNAs are synthesized as separate transcripts. D RNA Pol I transcription occurs in the nucleoplasm. E RNA Pol I transcription occurs in the cytoplasm. F rRNA gene clusters are known as nucleolar organizer regions.

3. Which one of the following statements about RNA Pol I transcription is false? A in RNA Pol I promoters the core element is 1000 bases downstream from the upstream control element (UCE). B upstream binding factor (UBF) binds to both the UCE and the upstream part of the core element of the RNA Pol I promoter. C selectivity factor SLl stabilizes the UBF-DNA complex. D SL1 contains several subunits including the TATA-binding protein TBP. E in Acanthamoeba there is a single control element in rRNA gene promoters. 4. Which two of the following statements about RNA Pol III genes are true? A the transcriptional control regions of tRNA genes lie upstream of the start of transcription. B highly conserved sequences in tRNA gene coding regions are also promoter sequences. C TFIIIC contains TBP as one of its subunits. D TFIIIB is a sequence specific transcription factor on its own. E in humans 5S rRNA genes are arranged in a single cluster of 2000 copies.

5. Which one of the following statements is true? A RNA Pol II only transcribes protein-coding genes. B the TATA box has a role in transcription efficiency but not in positioning the start of transcrip­hon. C TBP binds to the TAT A box. D Enhancers typically lie bp upstream from the start of transcription. 6. Which one of the following statements about general transcription factors is false? A TFIID binds to the T ATA box. B TFIID is a multi protein complex consisting of TBP and TAFIIs. C TBP is a common factor in transcription by RNA Pol I, RNA Pol II and RNA Pol III. D TFIIB stabilizes the TFIID-DNA complex. E TFIIE, TFIIH and TFIIJ associate with the transcription complex after RNA polymerase binding. F TFIIH phosphorylates the CTD.

THANK YOU !