Eukaryotic RNA polymerase II transcription cycle

chromatin opening

preinitiation complex formation

recycling

termination
initiation; regulation

elongation
promoter escape then pause

escape from pause

1
Fuda et al. (2009)
 Eukaryotic RNA polymerase II transcription

Meinhart & Cramer (2004) 430, 223-226

CTD: C-terminal Domain of large subunit

Tyr-Ser-Pro-Thr-Ser-Pro-Ser CTD can be phosphorylated on

specific Ser, Tyr and Thr residues
52 copies (human)
2
221 S16
 Different RNA processing
enzymes are recruited
sequentially by RNA
polymerase II CTD

3
221 S16
 Different patterns of CTD
phosphorylation help recruit the
correct mRNA processing 1
factors

3
Tyr-Ser-Pro-Thr-Ser-Pro-Ser

4
221 S16 B. Keith
 Dynamic phosphorylation patterns
of RNA polymerase II CTD during transcription
3’ cleavage and
5’ capping splicing poly(A) addition

(yeast)

Chromatin Immunoprecipitation (ChIP)

Tyr-Ser-Pro-Thr-Ser-Pro-Ser
5
221 S16 Heidemann et al., 2013
 Eukaryotic mRNA m7G 5’-cap:
Protection from 5’->3’ exonucleases

5’–5’ linkage

7-methyl guanine

6
 Phosphorylation of CTD tail of RNA polymerase II:
Ser5 phosphorylation of specific CTD repeats recruits capping factors

Required to prevent mRNA

cap degradation by 5’->3’
exonucleases
P

Tyr-Ser-Pro-Thr-Ser-Pro-Ser CTD: C-terminal Domain of large subunit

7
221 S16 B. Keith
 After 5’ capping, next processing event is pre-mRNA splicing, which is
associated with phosphorylation of CTD repeats on Ser2…

spl & poly(A)

P

Tyr-Ser-Pro-Thr-Ser-Pro-Ser CTD: C-terminal Domain of large subunit

52 copies (human)
8
221 S16 B. Keith
 Different patterns of CTD Ser2 phosphorylation are required for transcriptional
termination: cleavage & polyadenylation

Pattern of phosphorylated
repeats different from that
seen for splicing…

spl & poly(A)

P

Tyr-Ser-Pro-Thr-Ser-Pro-Ser CTD: C-terminal Domain of large subunit

52 copies (human)
9
221 S16 B. Keith
 Eukaryotic RNA polymerase II
mRNA 3’-end processing: cleavage & polyadenylation

CTD CPSF: Cleavage and Polyadenylation

Specificity Factor

CstF: Cleavage stimulation Factor

CPSF initially associated with Pol II, but

binds “AAUAAA” sequence in pre-mRNA
and recruits CstF and other factors.
Cleavage occurs ~30 bases downstream
(3’ to AAUAAA sequence
10
221 S16 Alberts et al. (2008) F 6-38 B. Keith
 Once cleaved, PAP (poly-A polymerase) adds ~200 “A” residues to the 3’ end of
the mRNA, which promotes stability, nuclear export, and translation. “polyA
binding protein” (PABP) binds and coats poly-A tail…

(CFI, CFII)

11
221 S16 Alberts et al. (2008) F 6-38 B. Keith
 PAP adds 3’ poly(A)tail, and poly(A) binding protein (PABP) binds to protect, and
also…

AAAAAAA

12
221 S16 Alberts et al. (2008) F 6-38 B. Keith
 Most eukaryotic mRNAs are circular…! PABP interacts with proteins
that bind the m7G 5’-cap to initiate mRNA translation into protein…

eIF4E: eukaryotic Initiation Factor 4E

Protein translation factors
eIF4G: eukaryotic Initiation Factor 4G

13
221 S16 Alberts et al. (2008) F 7-108 B. Keith
 Eukaryotic translation: circular polysomes

14
121.F15 Alberts et al. (2008) Figure 6-76
Prokaryotic translation: linear polysomes

Multiple ribosomes attached to an mRNA form a polyribosome.

15
 Dynamic phosphorylation patterns
of RNA polymerase II CTD during transcription
3’ cleavage and
5’ capping splicing poly(A) addition

(yeast)

Chromatin Immunoprecipitation (ChIP)

Tyr-Ser-Pro-Thr-Ser-Pro-Ser
16
221 S16 Heidemann et al., 2013
 Dynamic RNAP II phosphorylation patterns
are controlled by kinases and phosphatases

Phosphorylation pattern of CTD changes during elongation of pre-mRNA,

due to activity of kinases and phosphatases, which add or remove
phosphate groups from serine, threonine, or tyrosine residues
17
221 S16 Egloff et al., 2012
 Is there a RNA polymerase II CTD phosphorylation code?

There are many possible There are primary sequence

phosphorylation states differences among the repeats
18
221 S16 Egloff et al., 2012
 Different RNA processing
enzymes are recruited
sequentially by RNA
polymerase II CTD

These events typically occur co-

transcriptionally, but don’t have to.
They can occur independently of
the CTD, but recruitment of the
appropriate pre-mRNA processing
complexes by the CTD greatly
increases their efficiency…

19
221 S16 B. Keith
 Discovery of RNA splicing

* 1993 Nobel Prize in Chemistry 20

 Introns are removed from pre-mRNAs by the spliceosome, which catalyzes
two reactions that require specific sequences at the 5’ splice donor,
branchpoint, and 3’ splice acceptor sites…

* *

21
221 S16 B. Keith
 Splice junction consensus sequences

Alberts et al. (2008) F6-28

this alignment has to be perfect…!

22
221 S16 B. Keith
 Splice junction consensus sequences

invariant

23
221 S16 B. Keith