Structural Studies of RNA

Polymerase II Backtracking

UCL BIOC 0007 Workshop

24th October 2019
 Eukaryotic RNA Polymerases

Pol I rRNA

RNAP
Pol II mRNA Ribosome
Protein
DNA RNA

Pol III tRNA

mtRNAP
DNA RNA Protein
 Initiation

Termination Elongation
 Initiation Template
Promoter Promoter Initial RNA
complex strand
recognition opening catalysis
assembly positioning

RNA
Elongation Processive
separation
factor binding elongation
from template

Backtracking Cleavage

RNAP –
"Poly-A Tail
RNA cleavage template
formation"
dissociation
 Rpb1 Rpb2 Rpb3 Rpb4 Rpb5 Rpb6
Rpb7 Rpb8 Rpb9 Rpb10 Rpb11 Rpb12

RNA Template DNA Non-template DNA

 “Off-cycle processes”
l Priming
NTP l Mismatch recognition and
proofreading
Post
Translocation UV Damaged DNA
translocated l DNA Damage recognition

Pre Substrate l Translesion synthesis

translocated bound
l Transcript slippage

PPi l Template misalignment

RNA-DNA Product Catalysis
mismatch bound l Abortive Initiation
l Pausing/Backtracking
l Cleavage
 RNA Polymerase II
Pausing and Backtracking
 Wang (1995) Cell Galburt (2007) Nature

Paused vs. arrested RNAP

• Paused RNAPs eventually recover and continue transcribing
• Arrested RNAPs cannot continue elongation
• Arrested RNAPs have backtracked ~ 10nt.
• Backtracking is stimulated by
DNA sequence, or by protein
obstacles on the DNA.

• 3’OH disengages from active

site, inhibiting elongation

• Requires fwd translocation or

RNA cleavage to restore
elongation

Svestrup (2007) TIBS

The importance of backtracking

Nudler (2012) Cell

 Crystallising an backtracked Pol II complex

• Initiation factor
independent transcription + CTP
with CTP generated a + Pol II
Polycytosine RNA
– Electron density for 9nt of
backtracked RNA observed
 Backtracked transcript
binding locks translocation

Literature :
- Tailed template transcription arrests after synthesis of 13-17nt
- Arrested complexes release 7-14nt RNA upon TFIIS treatment
- Arrested complexes protect an extra 9nt of RNA compared to the elongation complex
 Pol II - backtracked RNA interactions

1. Conserved, 2. mostly non-base specific 3. TL interactions, 4. Gating Tyr, 5. Pocket, 6. Py preference

Catalytic residues
D290, E291

Pol II binding domain II

Catalytic domain III

Linker helix

DE to AA lethal
A Reactivation Complex : Backtracked Pol II-TFIIS
 Acknowledgements
UCL and Birkbeck College Rowan University, NJ, USA
Luis Miguel Diaz Santin Dmitry Temiakov
Emir Aciyan
NCI, Frederick, MD, USA
Andrew Wu Mikhail Kashlev
Lisa Watson Celine Walmacq
Daniel Saunders
Andy McGee LMU Munich/MPI Goettingen

George Cameron Patrick Cramer

Emma Kennedy Christoph Engel
Sarah Sainsbury