Polymerase I And Polymerase Iii Transcription

Lecture 3-RNA polymerase I and polymerase III transcription Homework question: Does pol II transcription take place in a particular region(s) of the nucleus?

Reviews of RNA pol I and pol III transcription

Moss, T. and Stefanovsky, V. Y. (2002) At the center of eukaryotic life. Cell 109, 545-548. Paule, M. R. and White, R. J. (2000) Transcription by RNA polymerases I and III. Nucl. Acids Res. 28, 1283-1298. Schramm, L. and Hernandez, N. (2002) Recruitment of RNA polymerase III to its target promoters. Genes Dev. 16, 2593-2620.

RNA pol I, pol II, and pol III transcribe different classes of genes Inhibition by α-amanitin RNA pol I •ribosomal RNA (rRNA), (e.g. 18S, 5.8S, and 28S)

>1 mg/ml (relatively insensitive)

RNA pol II •messenger RNA (mRNA), (i.e. protein-coding genes) •small nuclear RNA (snRNA), (e.g. U1 and U2) RNA pol III Structural or catalytic RNAs shorter than 400 bp •rRNA (5S) •snRNA (e.g. U6 and 7SK) •transfer RNA (tRNA)

0.01-0.05 μg/ml (relatively sensitive)

10-25 μg/ml (moderately sensitive)

Pol III Pol II

Pol I

In a proliferating eukaryotic cell: •18S, 5.8S, and 28S rRNA account for 35-60% of total nuclear transcription •5S and small nucleolar RNA (snoRNA) account for 10-20% of total nuclear transcription

RNA pol I transcription takes place in nucleoli

Nucleus

rRNA transcription takes place in fibrillar centres of nucleoli electron micrograph of a nucleolus

site of rRNA transcription site of rRNA processing site of assembly into ribosomes

Eukaryotes have several hundred identical genes encoding rRNA.

Spacer DNA (IGS) (18S, 5.8S, 28S)

“Miller spread” rRNA transcription in the egg of a spotted newt

•Up to 100 RNAs are synthesized simultaneously from a single gene

Generic organization of pol I transcription units rRNA repeats

• •

rRNA coding units are separated by intergenic spacers (IGS) IGS contain 1. a series of terminators (term), which are bound by TTF-1 2. a series of enhancers (function to assist stable complex formation) 3. a spacer promoter (SP), which have no known function 4. a proximal terminator (PT), which terminate transcripts from SP 5. the upstream promoter element (UPE, also known as the upstream control element (UCE or UE)), which is located , ~150-200 bp upstream of +1) 6. the promoter core (~50 bp upstream of the initiation site (+1)), which contains the only conserved rRNA promoter sequence element the ribosomal initiator (rInr)

Trans-acting factors that regulate rRNA transcription In mammals, 3 basal transcription factors have been identified: 1.

rRNA

yeast

2. 3.

SL1 or TIF-IB, the “selectivity” complex • TBP • TAF95/110 • TAF68/63 • TAF48 UBF, the upstream binding factor RNA pol I

Rrn3p bridges SL1 and pol I SL1 is then recruited along with pol I

UBF may recruit pol I through the polymerase associated factor 53 (PAF53) UBF can displace histone H1 (relieve repression) UBF, through its HMG1 boxes, can induce a chromatin-like structure

mammals

UBF binds the promoter first

DNase I footprinting experiment to examine the interaction of UBF and SL1 with the human rRNA promoter 1. 2.

UBF and/or SL1 were incubated with radiolabeled human rRNA promoter DNA DNase I was added to cleave exposed regions of DNA

3.

Samples were analyzed by electrophoresis

Result: SL1 cannot footprint on its own but enhances and extends the footprint of UBF Interpretation: SL1 may either directly contact DNA when complexes with UBF or may change the conformation of UBF so that it contacts a larger region of DNA

UBF induces a DNA supercoiling architecture called an enhancesome •The HMG (high mobility group) box is DNA-binding motif that induces severe bends in the target DNA but has little or no sequence specificity •UBF proteins have 4-6 HMG1 boxes •HMG1 boxes 1-3 of UBF (along with the N-terminal dimerization domain) are minimally required for activation of pol I transcription

5

UBF

HMG1 boxes

43 2 1

red-rRNA promoter yellow checkered box-upstream control element yellow box-core promoter blue spheres-HMG1 boxes

Pol I basal factors are regulated by protein-protein interactions and post-translational modifications Epidermal growth factor receptor

ERK1/2

Activation of rRNA transcription

green-activating interactions or modifications red-repressive interactions or modifications

phosphorylation of UBF disrupts the interaction with DNA and may enhance promoter clearance

rRNA promoters and trans-acting factors exhibit species specificity Examples: yeast

•There is very little sequence similarity between rRNA promoters from different species, but the general layout of functional promoter elements is highly conserved

mammals

•In yeast, distinct factors bind the UAF and the core promoter elements, whereas in mammals SL1 binds both elements

There are 3 types of RNA polymerase III promoters

Type 1: 5S Internal control region • A box (+50 to +60) • Intermediate element, IE (+67 to +72) • C box (+80 to +90)

Type 2: tRNA • •

A box (+8 to +19) B box (+52 to +62)

Type 3: Hs U6 Enhancer • Distal sequence element, DSE (-215 to -240) Promoter • Proximal sequence element, PSE (-65 to -48) • TATA box (-32 to -25)

All 3 types of genes terminate with a run of T residues

RNA pol III termination •A run of four or more T residues surrounded by G-C rich sequences on the non-coding strand constitutes the termination signal • This signal contributes to the efficiency of RNA pol III initiation and reinitiation (i.e. RNA pol III recycling)

•Four factors have been implicated in efficient termination and recycling 1. 2. 3. 4.

La protein/antigen- (role is not clear) binds the poly-U tail at the end of pol III transcripts NF1 polypeptides-mutation of NF1 binding sites downstream of the T’s results in read-through transcripts DNA topoisomerase I-suppresses read-through transcripts in a specialized system PC4-suppresses read-through transcripts in a specialized system

Biochemical identification of RNA pol III transcription factors Hela cell nuclear extract (competent for all pol III transcription in vitro)

Phosphocellulose column 100 mM KCl flowthrough

100-350 mM KCl step elution

350-600 mM KCl step elution

600-1000 mM KCl step elution

Fraction C TFIIIC

Fraction D SNAPC

Fraction A TFIIIA

Fraction B TFIIIB

•single protein

•TFIIB-related factor 1 (Brf1) •TFIIIC220/TFIIICα •Bdp1 •TFIIIC110/TFIIICβ •TATA binding protein (TBP) •TFIIIIC102/TFIIICγ •TFIIIC90/TFIIICδ •TFIIIC63/TFIIICε

•SNAP190 •SNAP50/PTFβ •SNAP45/PTFδ •SNAP43/PTFγ •SNAP19

Type 1 requires TFIIIA, TFIIIB, TFIIIC, and RNA pol III (17 subunits) Type 2 requires TFIIIB, TFIIIC, and RNA pol III Type 3 requires TFIIIB, SNAPC, and RNA pol III

antibody

no n co e n TF tro III l A

Antibody inhibition experiment to examine the requirement for TFIIIA

1.

2.

5S and tRNA genes were added to a Xenopus oocyte extract in the presence of radiolabeled nucleotides and (1) no antibody, (2) an irrelevant antibody, or (3) a TFIIIA antibody Reactions were analyzed for 5S and tRNA synthesis

Result: The TFIIIA antibody blocked 5S but not tRNA transcription Interpretation: TFIIIA is required for 5S but not tRNA transcription

Multiple TFIIIB complexes with promoter-selective activities

Human

Drosophila

TBP-related factor 1

red bars-strong protein-protein interaction blue bars-weak protein-protein interaction stippled line-no protein-protein interaction has been demonstrated

Recruitment of the common pol III factors: TFIIIB and RNA pol III

tRNA

snRNA: U6

Type 1: TFIIIA (ICR) > TFIIIC > TFIIIB > RNA pol III Type 2: TFIIIC (A and B boxes) > TFIIIB > RNA pol III Type 3: SNAPc (PSE) > TFIIIB-like (TATA) > RNA pol III 5S

Protein-DNA interaction (provides specificity)

green arrows-interactions of DNA-binding proteins with promoter elements blue arrows-protein-protein contacts among transcription factors purple arrows-protein-protein contacts between transcription factors and RNA pol III

Order of addition experiment to examine assembly of factors

1. 2. 3. 4.

TFIIIA (A), TFIIIB (B), and TFIIIC (C) were added sequentially to a 5S gene that was bound to cellulose After addition, unbound factors were washed away Finally, RNA pol III and radiolabeled nucleotides were added Reactions were analyzed for 5S synthesis

Result: 5S transcription only occurred when TFIIIB was added last Interpretation: TFIIIB needs the help of other factors to bind the 5S gene

Factors bound to internal promoters are displaced by RNA pol III type 2, tRNA

•TFIIIA is displaced by RNA pol III passage through the 5S gene

TFIIIA •a C2H2 zinc finger DNA-binding protein (9 fingers) •binds directly to the ICR of type 1 (5S) promoters (also binds 5S RNA) •little is known about how TFIIIA recruits TFIIIC

TFIIIB •functions to recruit RNA pol III •participates in opening of the transcription bubble bubble (Bdp1)

TFIIIC •recognition of promoter elements [either directly (for type 2 promoters) or with the help of TFIIIA (for type 1 promoters] •recruitment of TFIIIB •contributes to recruitment of RNA pol III

SNAPC •binds specifically to the PSE in type 3 (U6 and 7SK) promoters •the Pou-domain proteins STAF and Oct-1bind DSE sequences and contain activation domains for RNA pol III transcription (they also function in RNA pol II transcription through a different activation domain) DSE (OCTA) bound by Pou protein (Oct-1)

human U6 gene

positioned nucleosome

TFIIIB-like SNAPC

Summary

•RNA pol I and pol III are together responsible for the bulk of transcriptional activity in eukaryotic cells •Pol I transcription takes place in the nucleolus •In mammals, rRNA transcription takes place upon sequential recruitment of UBF, SL1, and pol I •rRNA transcription is regulated in a cell cycle and stress-dependent manner by post-translational modification of basal pol I factors and by protein-protein interactions with basal factors •There are 3 types of pol III genes that contain different promoter elements •TFIIIB and RNA pol III are recruited to the 3 types of promoters by different mechanisms

Homework question: Does pol II transcription take place in a particular region(s) of the nucleus?