Section M Transcription in Eukaryotes Molecular Biology Course.

Section M Transcription in Eukaryotes

•Molecular Biology Course

TopicsM1. The three RNA Polymerases: c

haracterization and function M2. RNA Pol I genes: the ribosomal

repeatM3. RNA Pol III genes: 5S and tRN

A transcriptionM4. RNA Pol II genes: promoters a

nd enhancersM5. General transcription factors a

nd RNA Pol II initiation


M1. The three RNA Polymerases: characterization and functionM1. The three RNA Polymerases: characterization and function

1. Eukaryotic RNA polymerases2. RNA polymerase subunits3. Eukaryotic RNA polymerase activities4. The CTD of RNA Pol II


M1-1: Eukaryotic RNA polymerases

M1-1: Eukaryotic RNA polymerases


1.The mechanism of eukaryotic transcription is similar to that in prokaryotes.

2.A lot more proteins are associated with the eukaryotic transcription machinery, which results in the much more complicated transcription.

Main Features of eukaryotic transcription


3. Three eukaryotic polymerases transcribe different sets of genes. The activities of these polymerases are distinguished by their sensitivities to the fungal toxin -amanitin ( 鹅膏菌素 ).

4. In addition, eukaryotic cells contain additional RNA Pols in mitochondria and chloraplasts.


Type Location Substrate -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

Nucleo-plasm

tRNAs, 5S rRNA,

U6 snRNA and other small RNAs

Moderately sensitive

Three eukaryotic polymerases

M1-2: RNA polymerase subunits

M1-2: RNA polymerase subunits


Each eukaryotic polymerase contains 12 or more subunits.– the two largest subunits are similar to each other and to the ’ and 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.

M1-3: RNA polymerase activities

M1-3: RNA polymerase activities


1.Transcription mechanism is similar to that of E. coli polymerase (How?)

2.Different from bacterial polymerasae, they require accessory factors for DNA binding.

M1-4: The CTD of RNA pol II

M1-4: The CTD of RNA pol II


1. 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.

2. The heptapeptide sequenc is: Tyr-Ser-Pro-Thr-Ser-Pro-Ser

3. This repeated sequence is known as carboxyl terminal domain (CTD)

4. 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 repeatsM2. RNA Pol I genes: the ribosomal repeats


1-2: Structure of the rRNA genes 1. Ribosomal RNA genes2. Role of the necleolus

3-6:RNA Pol I promoters & binding factors3. RNA Pol I promoters4. Upstream binding factor (UBF)5. Selectivity factor 16. TBP and TAFIs

7: Other rRNA genes


M2-1&2: Structure of the rRNA genes

1. Ribosomal RNA genes2. Role of the necleolus

M2-1&2: Structure of the rRNA genes

1. Ribosomal RNA genes2. Role of the necleolus


Ribosomal RNA Genes & nucleolus

1. A copy of 18S, 5.8S and 28S rRNA genes is organized as a single transcription unit in eukayotes. A 45S rRNA transcript (~13 000 nt long) is produced during transcription, which is then processed into 18S, 5.8S and 28S rRNA.

2. Pre-rRNA transcription units are arranged in clusters in the genome as long tandem arrays separated by nontranscribed spacer squences.

A single transcription unit

Tandem array

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 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”.

Christmas Tree Structures

M2-3~6: RNA Pol I promoters & binding factors3. RNA Pol I promoters4. Upstream binding factor (UBF)5. Selectivity factor 16. TBP and TAFIs

M2-3~6: RNA Pol I promoters & binding factors3. RNA Pol I promoters4. Upstream binding factor (UBF)5. Selectivity factor 16. TBP and TAFIs


RNA Pol I promoters1. Generally consists of a bipartite seque

nce in the region preceding the start site, including core element and the upstream control elements (UCE).

2. 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.

RNA Pol I promoters in human cells

Your text shows RNA Pol I promoter from another organism

Two ancillary factors (UBF & SL1) of RNA Pol I & their roles in transcripti

on initiation


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.

Selectivity factor 1 (SL1)1. Does not bind to promoters by itself 2. Binds to and stabilizes the UBF-DNA c

omplex.3. Interacts with the free downstream pa

rt of the core element.4. Recruit RNA Pol I to bind and to initiat

e the transcription.

Subunits of SL1

SL1 consists of 4 proteins. 1. TBP (TATA-binding protein): a factor a

lso 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.

2. 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

The initiation complex proposed in your text book

UBF

UBFRNA Pol I

TBP

TAFIs

TAFIs

TA

FIs

It is not known which representation one is more accurate.

Other rRNA genes (simple)In a simple eukaryote, Acanthamoeba, the rRNA genes have only one control element (promoter) around 12-72 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 transcriptionM3. RNA Pol III genes: 5S and tRNA transcription


1. RNA polymerase III2. tRNA genes3. 5S rRNA genes4. Alternative RNA Pol III promoters5. RNA Pol III termination

M3-1. RNA Pol IIIM3-1. RNA Pol III


1. Contains at least 16 or more subunits2. Is located in nucloplasm3. Synthesizes the precursors of 5S rRNA, t

he tRNAs and other small nuclear and cytosolic RNAs

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).

M3-2. tRNA genesM3-2. tRNA genes


1. The initial transcripts of tRNA genes need to be processed to produce the mature tRNA.

2. 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 TC-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

M3-3. 5S rRNA genesM3-3. 5S rRNA genes


1. Tandemly arranged in a gene cluster. (In human, there is a single cluster of around 2000 genes.)

2. Transcription control regions (promoters) are organized similar to those of tRNA, except that C box is in place of B box. C box: +81-99 bp; A box: +50-65

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.

TFIIIA

TFIIIC

TFIIIB

Pol III

M3-4. Alternative RNA Pol III promoters

M3-4. Alternative RNA Pol III promoters


Many RNA Pol III genes also rely on upstream sequences for regulation of their transcriptione.g. U6 snRNA and Epstein-Barr virus

1. Use only regulatory genes upstream from their transcription start sites.

U6 snRNA1. The coding region contains a characteristic

A box that is not required for transcription. 2. The upstream sequence contains sequences t

ypical 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

M3-5. RNA Pol III terminationM3-5. 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’

M4. RNA Pol II genes: promoters and enhancersM4. RNA Pol II genes: promoters and enhancers


1.RNA Pol II2.Cis-acting elements• Promoters• Upstream regulatory elements• Enhancers

M4-1. RNA Pol IIM4-1. RNA Pol II


1. located in nucleoplasm2. catalyzing the synthesis of the m

RNA precursors for all protein-coding genes.

3. RNA Pol Ⅱ-transcribed pre-mRNAs are processed through cap addition, poly(A) tail addition and splicing.

M4-2. PromotersM4-2. Promoters


• Most promoters contain a sequence called the TATA box around 25-35 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.

M4-3. Upstream regulatory elements

M4-3. 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 100-200 bp from the promoter, and plays an important role in ensuring efficient transcription.

M4-4. EnhancersM4-4. 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 transcription factors and RNA PolⅡ initiationM5. General transcription factors and RNA PolⅡ initiation


1. RNA Pol II basal transcription factors2. TFIID (TBP)3. TFIIA4. TFIIB and RNA Pol binding5. Factors binding after RNA Pol.6. CTD phosphorylation by TFIIH7. The initiator complex

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

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

TBP

DNA

TBP: 3. Has a saddle structure with an overall dyad symmetry.

Outer surface (with ?)

Inner surface (with ?)

TBP causes DNA bending

TBP

45o Kink

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

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

4-1 TFIIE binding•Necessary for transcription

4-2 TFIIJ, TFIIH binding•Necessary for transcription

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

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.

Thanks

Section M Transcription in Eukaryotes Molecular Biology Course.

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