Molecular Biology II Eukaryotic Transcriptional

Machinery

Eukaryotes have 3 different nuclear RNAPs:

 RNAPI transcribes 5.7S, 18S and 28S rRNAs (nucleolus)
 RNAPII transcribes all mRNAs (nucleus)
 RNAPIII transcribes 5S rRNA, all tRNAs and other small RNAs (nucleus)

Mitochondria have their own RNAP (mtRNAP), as do chloroplasts (they have their own genomes)

There is a similarity between the bacterial core RNAP, and eukaryotic RNAPIIs

RPB1 & RPB2 are similar to β’ & β

RPB3, RPB10, RPB11 & RPB12 are similar to the α2 homodimer
RPB6 is similar to ω

The core is similar, with the addition of peripheral subunits, that are not involved in promoter
recognition

Bacterial RNAPs in the presence of sigma factors can recognise promoters without the help of any
other transcription factors.

Eukaryotic RNAPs have more subunits, but still have no obvious sequence specific binding
activities.
They require the assistance of additional factors (basal factors) to recognise promoter elements
(TFIIA/B/D) and for the formation of the transcription bubble around the transcript start position
(TFIIE/H)

Lecture 19 [Page 1]
Molecular Biology II Eukaryotic Transcriptional
Machinery

Eukaryotic RNAP recognises the promoter via a series of protein-protein interactions

DNA is not recognised directly by the RNAP

The assembled initiation complex is the same for every RNAPII-transcribed gene, and only gives
low (basal) levels of unregulated transcription
Gene specific transcription factors are required to give more regulated transcription

The basal initiation complex, containing

RNAP, binds around +1
Basal TFs position RNAP correctly

Proximal gene-specific transcription

factors bind around 1000bp upstream
They stimulate transcription

Distal gene specific TFs (enhancers)

are 50kb+ upstream. DNA loop forms,
bringing enhancer near to RNAP. DNA
within the loop is wound up as
chromatin.

Distance from start site does not

correlate with the actual distance of
TFs as they are all brought together at RNAP.

Promoter Recognition:

Some viruses have very strong promoters, as they need to compete with host genes for
transcription machinery (e.g. adenovirus ‘major late’ promoter)
Their promoters are almost identical to the idealised model of a promoter (below)

A TA rich sequence embedded between GC rich sequences

BRE – TFIIB Recognition Element. The main TFIIB contact point.

A cis-regulatory element found immediately upstream of the TATA box. 7 nucleotides long

Lecture 19 [Page 2]
Molecular Biology II Eukaryotic Transcriptional
Machinery

Cis-regulatory element – a region of DNA / RNA that regulated expression of genes located on
the same molecule of DNA / RNA
Trans-regulatory element – modify expression of genes distant from the gene that was originally
transcribed to create them

TFIID:

The TATA box is specifically recognised by TFIID

TFIID is a multiprotein complex consisting of
TATA-Binding Protein (TBP)
 Can sequence-specifically bind to the TATA box on its own
 Horseshoe shaped
 Causes a conformational change in DNA on binding, resulting in a 90 kink
 This allows RNAP to bind more directly to DNA
TBP-Associated Factors (TAFs) ~12 of these
Non-covalent interactions hold the subunits together
TAFs bind to other promoter elements if TBP cannot bind to TATA box

TFIIA/B stabilise TFIID binding – DNA bending is energetically unfavourable

TF complexes were detected using EMSA

(Electrophoretic Mobility Shift Assay)

Samples are run on native gels (no urea / SDS) to keep

proteins in natural conformation

The technique allows us to determine whether s protein has

bound to radiolabelled DNA (gel is later exposed to X-ray
film)

Other Promoter Elements:

Only ¼ RNAPII-transcribed genes have TATA boxes

There are 2 more consensus motifs that maybe present

 Initiator Element (IE) – makes contact with transcription start site
 Downstream Promoter Element (DPE)
TAFs recognise these additional sequence elements

Lecture 19 [Page 3]
Molecular Biology II Eukaryotic Transcriptional
Machinery

+1 is always A/G
TATA box position is more variable in eukaryotes than prokaryotes
There is some sequence variation in eukaryotic TATA boxes
DPE is more common than TATA box
Most eukaryotic genes have a combination of these elements
In promoters that lack a TATA box, TFIID is recruited by a sequence-specific contact with the
initiator or DPE elements

TFIID

TFIIA

TFIIB

TFIIF + RNAPII

TFIIE

TFIIH

The resulting complex (basal machinery) is the

minimal functioning complex for basal transcription

Lecture 19 [Page 4]
Molecular Biology II Eukaryotic Transcriptional
Machinery

TFIIF binds to RNAP and facilitates its delivery to the TFIID-TFIIB-DNA complex on the promoter
TFIIE stimulates TFIIH, the pair have critical responsibilities in transcription
 I) Phosphorylation of RNAPII, making the enzyme elongation competent
o TFIIH has kinase activity
 II) Promoter melting via DNA helicase mechanism
 Promoter clearance

I) Phosphorylation of RNAPII
RBP1 is the largest subunit in RNAPII
It has a long C-terminal tail consisting of 26 (yeast) or 52 (human) repeats of a 7nt sequence
YSPTSPS
The sequence is phosphorylated by TFIIH kinase
S (serine) residues can be phosphorylated
P (proline) residues disrupt 3D structure

Hypophosphorylated ( phosphorylation) – CTD is associated with the initiation complex

Hyperphosphorylated ( phosphorylation) – CTD with elongation-competent RNAPII

The level of CTD phosphorylation regulates a number of other processes associated with
transcription:
 5’ capping
 Assembly of spliceosomes
 Binding of the cleavage / polyadenylation complex

II) Promoter Melting by TFIIH

TFIIH melts the dsDNA around the transcription start site to form the transcription bubble
It does this by twisting DNA at the start site
The melted promoter is intrinsically unstable (45 seconds half-life)
Prokaryotic open complex on the other hand is stable
If transcription initiation does not occur in this time then the melted promoter configuration can
only be held open by continual ATP hydrolysis
This is an important control point for regulating the rate of initiation of transcription as initiation can
only occur if the transcription bubble is present

In bacteria, the -10 region initiates the formation of transcription bubble

Lecture 19 [Page 5]