1

Three types of RNA polymerase in eukaryotic nuclei

Type Location RNA synthesized Effect of α-amanitin

I Nucleolus Pre-rRNA for 18, 5.8 and 28S rRNAs InsensitiveII Nucleoplasm Pre-mRNA, some snRNAs Sensitive to 1 µg/mlIII Nucleoplasm Pre-tRNAs, 5S rRNA, some snRNAs Sensitive to 10 µg/ml

(Also- Organelle RNAPs in Mitochondria and Chloroplasts. Encoded by organellegenomes. Similar to bacterial RNAPs.)

• α-amanitin from Amanita Phalloides binds tightly to RNA Pol II and blockstranscriptional elongation.• RNA Pol I transcribe 1 gene at ~200 copies. The gene for the 45S pre-rRNA ispresent in tandem array.• RNA Pol II transcribe ~25,000 genes;• RNA Pol III transcribe 30-50 genes at variable copy numbers.

Subunit composition of eukaryotic RNA polymerases•All three yeast polymerases have fivecore subunits that exhibit some homologywith the β, β‘, α and ω subunits in E. coliRNA polymerase.

•RNA polymerases I and III contain thesame two non-identical α-like subunits,whereas polymerase II has two copies of adifferent α-like subunit.

•All three polymerases share four othercommon subunits. In addition, each RNApolymerase contains three to seven uniquesmaller subunits.

•The largest subunit (1) of RNApolymerase II also contains an essential C-terminal domain (CTD). 27 (yeast) to 52(human) copies of (YSPTSPS).

•Phosphorylation of CTD is important fortranscription and RNA processing.

2

Comparison of 3-D structures of bacterial and eukaryotic RNA polymerases

(subunits 4and 7 aremissing)

Cyan: alternating β-turns; Pink: extended regions.

3

Core Promoter Elements

Many genes, which are transcribed at low rates (e.g. genes encoding the enzymesrequired for basic metabolic processes required in all cells, often called“housekeeping genes”) do not contain a TATA box or an initiator. Most genes of thistype contain a CG-rich region, or CpG island, of 20-50 nucleotides within ~100 basepairs upstream from the start site. Transcription of these genes can begin at any one ofmultiple possible sites over an extended region.

RNA Pol II is recruited to corepromoter with the help from

multiple general transcriptionfactors.

In vitro stepwise assembly of theRNA Pol II pre-initiation complex(PIC) at core promoter for basal

transcription

4

Basal (‘General’) Transcription Factors for RNA Polymerase II

Total: 43-44 polypeptides and over 2 million daltons.

TBP (TATA-box binding protein)

•Conserved C-terminal domain of 180amino acids.•A monomer with a saddle-shapedstructure; the two halves show anoverall dyad symmetry but are notidentical.•Binds multiple transcription factors(TAFs, TFIIB and TFIIA).•Binds in the minor grooveand significantly bends DNA.

5

Eukaryotic transcription cycle

Only theunphosphorylatedRNA Pol IIenters PIC.

The TFIIH complexhas both helicaseand kinase activitiesthat can unwindDNA andphosphorylate theCTD tail of RNAPol II.

Release ofTFIIE and thenIIH during thesynthesis of theinitial 60-70nt.

CTD

PICassembly

Pol II

CTD

Pol II Pol II555

5

TFIIH

Promoterclearance& pausing

for capping

CycT1CDK9

P-TEFb

Releasefrom

pausing

Pol II

Productiveelongation

Pol IIPol II

CTDCTD

FCP1Recycling

5’ cap

2

555

5222

2

555

5222

2

555

5222

RNA+1

CTD heptapeptide repeats: 27-52 x (YS2PTS5PS)

Phosphorylation states of Pol II CTD during transcription cycle

6

Cis-acting control elements

(a) Genes of multicellular organisms contain both promoter-proximal elements andenhancers (collectively referred to as cic-acting control elements) in addition to corepromoter element(s).

(b) Enhancers function in a distance, position and orientation-independent manner. Longdistance interactions are achieved by forming looped DNA.

(c) Most yeast genes contain only one regulatory region, called an upstream activatingsequence (UAS), and a TATA box, which is ≈90 base pairs upstream from the start site.(Also note: many yeast genes do not contain introns).

(d) In multicellular organisms, one standard promoter-proximal element often located in theCpG island is a GC-box (GGGCGGGC) recognized by the ubiquitous transcriptionalactivator Sp1.

DNA affinity chromatography for purification of Sp1

7

Analyses of affinity-purified Sp1 protein

DNase I footprint on SV40 promoter

SDS-PAGE/ silverstain

Sp1

NaCl

Lane 2 containstotal cell proteinsprior to affinitypurification;Lanes 3&4 containpurified Sp1protein washed offthe affinity column.

6 x

GC

-box

es

“Gel shift”: electrophoretic mobility shift assay (EMSA) forstudying the interaction of Sp1 with GC-box

Free DNA probe*

*Protein-DNA complex

1. Prepare labeled DNA probe2. Bind protein3. Native gel electrophoresis

Advantage: sensitive Disadvantage: requires stable complex;little information about where protein isbinding on DNA

ON: protein mixture loaded onto an ion-exchangecolumn.Fraction 1-22: fractions eluted from the column withincreasing salt concentrations.

Sp1GC-box

8

In vitro transcription assay to measure Sp1 activity

•The adenovirus DNAtemplate used here does notcontain any Sp1-binding sites(GC-box) and is therefore usedas a negative control.

•In vitro transcriptionreactions contain templateDNA, labeled ribonucleosidetriphosphates, and purifiedgeneral transcription factorsand RNA Pol II. Purified Sp1is added to the reactionsindicated with “+”.

In vivo assay for transcription factor activity

co-transfection

• Host cells should lack thegene encoding protein Xand the reporter protein.

• The production ofreporter-gene RNAtranscripts or the activityof the encoded protein canbe assayed.

• If reporter-genetranscription is greater inthe presence of the X-encoding plasmid, then Xis an activator; iftranscription is less, thenX is a repressor.

Reporter-gene products

9

Identification of promoter-proximal cis-acting controlelements upstream of a eukaryotic gene (part 1)

Identification of promoter-proximal cis-acting controlelements upstream of a eukaryotic gene (part 2)

10

Graphic representation of the conservation of DNA sequences within acorresponding region in five different genomes reveals a region of ~500 bp ofnon-coding sequence that is conserved from fish to human.

Computer-assisted search for enhancers

The Production of DNA Microarrays and Their Use inMonitoring Global Gene Transcription