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

Section M: Transcription in Eukaryotes Yang Xu, College of Life Sciences

Section M Transcription in Eukaryotes

M1 The three RNA polmerases: Characterization and functionM2 RNA pol. I genes: The ribosomal repeatM3 RNA pol. III genes: tRNA and 5S ribosomal repeatM4 RNA pol. II genes: Promoters and enhancersM5 General transcri. factors and pol. II initiation


M1 The three RNA polmerases:Characterization and function

• Eukaryotic RNA polymerases

• Eukaryotic RNA polymerases activities

• RNA polymerase subunit


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.


Eukaryotic RNA polymerases

Transcription

for

Location

in

Sensitivity to

-amanitin

RNA pol Imost of rRNA genes

nucleoli insensitive

( － )

RNA pol IImRNA and some snRNA

nucleoplasm very sensitive (+ +)

RNA pol IIItRNA and

5S rRNA

nucleoplasm moderately sensitive (+)


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 II RNA 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


M2 RNA pol. I genes: The ribosomal repeat

• Ribosomal RNA genes• Promoter of RNA pol. I• Upstream binding factor• Selectivity factor 1• TBP and TAFIs• Other rRNA genes


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 13 000 nt long.

18S 28S

5.8S

Singletranscr. 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 Eukaryotes Yang Xu, College of Life SciencesSection O: RNA Processing and RNPs. 华中师范大学生科院杨旭

Eukaryotic ribosomesThe complete mammalian (rat) 80S ribosome is composed of one

large 60S subunit and one small 40S subunit. The 40S subunit contains an 18S rRNA molecule and about 30 distinct proteins. The 60S subunit contains one 5S rRNA, one 5.8S rRNA, one 28S rRNA and about 45 proteins

5 S(120 bases)

+

ProteinsL1, L2, L3….L45

Sub-units

60 S

18S(1900 bases)

+

S1, S2, S3….S30

r RNA

40S

28S 5.8S(4800+160bases)

80 S


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 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.

+1

Pre-rRNA gene

-100

Core element+6-31

UCE50-80 bp


• UBF• UBF-1 + UCE• UBF-2 + Part of core promoter• Two UBF interaction causing DNA to form a loop

3 TAF1 TBP

SL1

Upstream binding factor (UBF)

UCE core promoter UBF UBF


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.


Selectivity factor 1SL1: 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 TAFIsSL1 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);• TAFIs: 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 TAFIs.

3 TAF1 TBP


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


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.


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 TC-loop of tRNA )

ACC

anticodon loop

5’

TC-loop acceptor arm

D loop

V loop

A box B box


TFIIIC

Fig. 1. Initiation of transcription at a eukaryotic tRNA promoter

A box B box

TFIIIC

B”TBP

BRF

RNA pol III

B”TBP

BRF

TFIIIB

RNA pol III


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


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 81-99 bp downstream from the transcription start site.

• A box: A second sequence termed the A box around bases +50 to +65 is also important.


TFIIIC

Fig. 2. Initiation of transcription at a eukaryotic 5S rRNA promoter

TFIIICRNA pol III

B”TBP

BRF

TFIIIB

TFIIIA

A box C box

+81 +99+50 +65

TFIIIA RNA pol III B”TBP

BRF


Promoters of RNA pol. III

1. Eukaryotic tRNA promoter

2. Eukaryotic 5S rRNA promoter

A box B boxTATA box +1

-30 -23

A box C boxTATA box +1

-30 -23


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.


M4 RNA pol II genes: Promoters and enhancers

• RNA polymerase II

• Promoters of RNA pol. II

• Upstream regulatory elements (URE)

• Enhancers


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

82 97 93 85 63 (37) 83 50 (37)

1-4Kb -70 -30 -1C +1A

GC island CAAC box TATA box Cap

Enhancer UPE core promoter Promoter (basic factor)

-270 -170

URE


Upstream regulatory elementsThe 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

100-200 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


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


RNA Pol II CTDF

RNA Pol IIF

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

TAFIIs

ATFIIA

BB TFIIB

E

E

H

H

J

J

mRNA

TBP

TAFIIsTFIID

RNA Pol II CTDF

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 IITFII 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 E

H J

3×3


TATA +1 Winds minor groove

TFII A TFII D

Pre -TIC

TFII F RNApol IITFII B

Basic TIC

conclusion

TIC: Transcriptional Initiation Complex


mRNA

Promoter clearance

Transcription starting

complete TIC

E

HJ

E H B D AJ

9 － 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 26XDrosophila 44X repeat unit of 7 aa / in CTDRat & Human 52X

IIO (240kd) IIA (220kd) IIB (180kd)

With all Without all Basic form

RNApol easy to leaves Promoter for elongation

--CTD-end construction:

7aa repeats & high frequency phosphorylation

Tyr—SerP—Pro—ThrP—SerP—Pro—SerP


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