RNA Polymerase II

and Transcription
Initiation
 RNA polymerases
Cellular RNA polymerases are large multisubunit complexes:

Bacteria: single RNAP a2bb’w

Eukaryotes: three distinct RNAPs:
- RNAPI 5.7S/18S/28S rRNA
- RNAPII mRNAs, some snRNAs
- RNAPIII 5S rRNA, tRNAs, other small RNAs
Archaea: one eukaryotic-like RNAP

Mitochondrial polymerases are single subunit enzymes and

resemble viral RNA polymerases (eg. bacteriophage T7 RNAP)
Chloroplast RNAPs resemble the bacterial enzymes (symbiontic
origin of chloroplasts!)
 Evolution of RNAPs
Archaea Eukaryotes

Bacteria

LUCA
 Eukaryotic RNA polymerase II
• All eukaryotic RNAPIIs characterized so far contain 12 different subunits (RPB1-
RPB12)
• The two largest subunits, RPB1 and RPB2, contain the catalytic center and are
homologous to the
bacterial b and b’ subunits.
• Four subunits (RPB3, RPB10, RPB11 and RPB12) serve as an assembly platform.
• Five subunits (RPB5, RPB6, RPB8, RPB10, RPB12)
are common to all three eukaryotic RNAPs.
• The largest subunit (RPB1) has a long C-terminal
tail (CTD) constituted by up to 52 heptapeptide repeats (consensus: YSPTSPS).
 Eukaryotic RNAPII Structure
The 3D structure of the S. cerevisiae RNAPII has been determined at
atomic resolution.
This is the structure of the
10-subunit core, lacking
subunits RPB4 and RPB7.

Subunits RPB3/11 are

structurally similar to the
bacterial a dimer.
Subunit RPB6 is similar
to the w subunit.
 Clamp Opening – Frontal View

Superimposition of
Minor structural
deviations two yeast RNAPII
structures with
different Clamp
positions suggests a
simple, highly
localized hinge
mechanism
(involving the
PDB# 1I6H “Switch” regions)

PDB# 1I3Q
 Electrostatic Potential

negative positive neutral

RNA polymerases have very asymmetric charge distributions: the internal
channel (which binds nucleic acids) is highly positively charged, while the
external surface is highly negatively charged.
Cramer et al. (2001). Science 292, 1863-1876.
 The Catalytic Center

Clamp
Nascent Template
RNA DNA strand
Transcript
Non-coding
DNA strand
Downstream DNA
Active Site
Metal ‘A’

Bridge Helix

Klug (2001). Science 292, 1844-1846.

 Transcription
Nucleic Acid Substrate

Direction of
RNA Exit Path

Path in RNAP
The template DNA strand
bends by ~90 degrees in
the active site

Active Site

Newly
inserted NTP Direction of Transcription
Westover, K.D., Bushnell, D.A. and Kornberg, R.D. (2004). Structural basis of transcription: nucleotide selection
by rotation in the RNA Polymerase II active center. Cell 119, 481-489.
 Strand Separation Events

• The template DNA strand needs to be separated from the non-

template strand
• The template DNA strand enters the active site and a
complementary RNA is synthesized to create a short (~10 bp) DNA-
RNA hybrid
• The RNA needs to be ‘stripped’ from the DNA template strand
• The DNA template and DNA non-template strands need to be
reunited into double-stranded DNA
 NTP Entry into Active Center: The Classic View

• NTPs diffuse to the

active center of RNA
polymerase through a
funnel-shaped channel
(CH1) to bind to a
single position of the
exposed template
CH1 DNA strand

Batada, N.N., Westover, K.D., Bushnell, D.A., Levitt, M., and Kornberg, R.D. (2004). Diffusion of nucleoside triphosphates and role of the entry site to
the RNA polymerase II active center. Proc. Natl. Acad. Sci. USA 101,17361-17364.
 NTP Entry into Active Center: Latest View
• NTPs diffuse to the active center
of RNA polymerase not only
through CH1 to bind to a single
position of the exposed template
DNA strand, but also through an
additional channel, CH2, so that
they are able to base-pair in
parallel to multiple positions (i+2,
i+3, i+4) of the DNA template
strand

Génin, N.E., and Weinzierl, R.O. (2020). Nucleotide loading modes of human RNA polymerase II as deciphered by molecular simulations. Biomolecules 10:
E1289.
 The Translocation Mechanism

Bridge Helix
Metal ‘A’
NTP
RNA Trigger Loop

DNA Template Strand

Side View Top View

 Bridge Helix-Trigger Loop
Translocation

• Conformational changes in the Bridge Helix (green) and Trigger Loop (blue) move the
DNA-RNA hybrid through the active site and create space for new NTPs to be inserted

Kaplan, C.D., Kornberg, R.D. (2008). A bridge to transcription by RNA polymerase. J. Biol. 7, 39.
Tan, L., Wiesler, S., Trzaska, D., Carney, C. and Weinzierl, R.O. (2008). Bridge helix and trigger loop perturbations generate superactive RNA
polymerases. J. Biol. 7, 40.
Full-length
Transcripts
The Abortive
Transcription Cycle
• During transcription initiation, RNA
polymerases repeatedly produce a number
of short (2-11 nucleotide) transcripts
before they engage in successful
transcription (‘promoter escape’)
• If they fail to escape the promoter, the
short transcript is ‘aborted’ and
transcription starts once again from the +1
Abortive position
Transcripts
• Depending on the promoter, only 1-5% of
initiated transcripts will eventually end up
as full-length RNAs
• 95-99% of initiated transcripts will be aborted!
 Promoter Scrunching

Kapanidis, A.N., Margeat, E., Ho, S.O., Kortkhonjia, E., Weiss, S., and Ebright, R.H. (2006). Initial
transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314, 1144-1147.
 FRET-Probe

Non-template Strand Scrunch

FRET-Probe #1

Template Strand Scrunch

FRET-Probe #2
 RNA Exit from
RNAP

NTPs

Westover, K.D., Bushnell, D.A., and Kornberg, R.D. (2004). Structural basis of transcription: separation of RNA from DNA by RNA polymerase II. Science
303,1014-1016.
 TFIIB Recruitment of RNAPII
• The N-terminal Zn-ribbon domain of TFIIB interacts with the RNAPII
(via the ‘dock domain’ on RNAPII)
• Next, DNA is then opened with the help of the ‘B-linker’
• The DNA template strand slips into the cleft and is scanned for the transcription start site
with the help of the ‘B-reader’ that approaches the active site
• Synthesis of the RNA chain and rewinding of upstream DNA displace the B-reader and
B-linker, respectively, to trigger TFIIB release and elongation complex formation

Kostrewa, D., Zeller, M.E., Armache, K.J,, Seizl, M., Leike, K., Thomm, M., and Cramer, P. (2009). RNA polymerase II-TFIIB structure and mechanism
of transcription initiation. Nature 462, 323-330.
Kostrewa, D., Zeller, M.E., Armache, K.J,, Seizl, M., Leike, K., Thomm, M., and Cramer, P. (2009). RNA polymerase II-TFIIB structure and mechanism of
transcription initiation. Nature 462, 323-330.

http://naturedocumentaries.org/10545/movie-rna-polymerase-ii-transcription-cheung-cramer-2012/
 TFIIB Affects Initiation and
Promoter Escape
• While the components necessary to achieve specific initiation have
been known for some time, the transition from the initiation
complex to the transcript elongation complex is not well understood
• This transition must involve the disruption (“promoter escape”) of
numerous protein-protein contacts of RNAPII with initiation factors
and promoter elements

Pal, M., Ponticelli, A.S. and Luse, D.S. (2005). The role of the transcription bubble and TFIIB in promoter clearance by RNA polymerase II. Mol. Cell 19,
101–110.
The CTD Domain …
The C-terminal Domain (CTD)

The largest subunit of RNAPII has a long C-terminal tail

consisting of
26 (yeast RNAPII) to 52 (human RNAPII) repeats of a 7-residue
motif
(consensus: YSPTSPS)

Chapman, R.D., Heidemann,M., Hintermair ,C., and Eick, D. (2008). Molecular evolution of the RNA polymerase II CTD.
Trends Genet. 24, 289-296.
 The RNAPII CTD-Domain
• The presence of phosphorylatable amino acid residues in the
CTD heptad repeat provides an opportunity for additional
regulation
• Y (tyrosine), S (serine) and T(threonine) are phosphorylated by TFIIH
kinase
• The CTD is only present in eukaryotic RNAPII, not in
RNAPI, III, IV and V!
• Prokaryotic (bacterial, archaeal) RNAPs also lack an equivalent structure
 The C-terminal Domain (CTD)

Y (tyrosine), S (serine) and T(threonine) are phosphorylated by TFIIH kinase

Hypophosphorylated CTD is associated with the initiation complex,
hyperphosphorylated CTD with the elongation complex

The degree of phosphorylation of the CTD regulates a number of processes

associated with transcription: 5’ capping, the assembly of spliceosomes and the
binding of the cleavage/polyadenylation complex

Chapman, R.D., Heidemann,M., Hintermair ,C., and Eick, D. (2008). Molecular evolution of the RNA polymerase
II CTD. Trends Genet. 24, 289-296.
 ‘Open-Complex’ Formation
• Formation of the transcription bubble (‘open complex’) involves
localized ‘melting’ of a region of double-stranded (helical) DNA and
various conformational changes in the RNAP
• Melting of DNA is aided by the TFIIH helicase-activity
• The template-strand is loaded into the active site of the RNAP, the
non-template strand bypasses the catalytic center
• This process is ATP-dependent (energy-driven) in eukaryotic RNAPII,
but not in archaeal, bacterial or eukaryotic RNAPI and RNAPIII
 TFIIH: A Multisubunit Complex
• The TFIIH core forms a crescent-shaped
complex spanning from Ssl2 to Rad3
• Ssl2 binds downstream DNA consistent
with its role in DNA opening
• Ssl2 uses ATP hydrolysis to translocate on
DNA. If the Ssl2 location is fixed, Ssl2
action results in a reeling of DNA into the
active centre

Schilbach, S., Hantsche, M., Tegunov, D., Dienemann, C., Wigge, C., Urlaub, H.
& Cramer, P. (2017). Structures of transcription pre-initiation complex with TFIIH
and Mediator. Nature 551, 204–209.
 Transcription Bubble Opening

Schilbach, S., Hantsche, M., Tegunov, D., Dienemann, C., Wigge, C., Urlaub, H. & Cramer, P. (2017). Structures of transcription pre-initiation
complex with TFIIH and Mediator. Nature 551, 204–209.
TFIIH Complexed With RNAPII
RNAPII and mRNA Processing
RNAPII
transcription
and pre-mRNA
processing are
coordinated events

Capping, splicing
and polyadenylation
occur during
transcription and
RNAPII (and the
CTD particularly)
plays a role in the
splicing regulation of
these events
polyadenylation
capping